Reagents and method for assaying biological samples



United States Patent 3,413,198 REAGENTS AND METHOD FOR ASSAYINGBIOLOGICAL SAMPLES Alfred Deutsch, Los Angeles, Calif., assignor toCalbiochem, Los Angeles, Calif., a corporation of California No Drawing.Continuation-impart of application Ser. No. 320,004, Oct. 30, 1963. Thisapplication June 30, 1966, Ser. No. 561,757

14 Claims. (Cl. 195-1035) ABSTRACT OF THE DISCLOSURE Substantiallyanhydrous, solid assay materials for the determination, inter alia, ofglucose, adenosine triphosphate, glucose-6-phosphate dehydrogenase, andcreatine phosphokinase are rendered storage stable by the presence ofcertain polyhydric compounds, preferably mannitol, sorbitol, lactose orpolyvinyl alcohol.

'This invention is a continuation in part of my copending applicationSerial No. 320,004, filed October 30, 1963, now abandoned.

The present invention relates to processes and compositions forpreparing reagent mixtures for detecting and measuring the presence ofcertain components in a biological sample, It also relates to the novelreagent mixtures.

In the clinical diagnosis of certain pathological conditions, it isfrequently valuable to know the amount of activity or the quantity ofcertain substances present in a specimen of a biological or other fluid,or tissue. One of the more effective means that has been proposed formaking assays of such specimens is to provide a liquid reagent whichcontains one or more biological components. When a given reagent ismixed with the specimen, the components are effective to cause anezymatic reaction that involves the unknown substance. By observing thisreaction, it is possible to determine the quantity or amount of activityof the unknown originally present.

Since such reagents contain one or more biological components such asenzymes, coenzyrnes and/or substrates, etc., the reagent has inherentlybeen of a very unstable nature and has very little if any shelf life. Toinsure the reagent being at optimum strength it must be prepared at orimmediately prior to the time the assay is made. In adidtion, heretoforethe various components such as the enzymes, coenzyrnes, substrates,etc., included in the reagent have been very unstable. To insure thesecomponents being at their optimum it has been necessary for thecomponents to be stabilized in a concentrated form.

When it has been desired to make a biological assay of the present type,a kit containing the several different components which may be dry, orin solutions, has been obtained. If the components are in a dry form,aqueous solutions are formed, and maintained separately until just priorto use.

The various components for the reagent are present in separatecontainers and maintained separated from each other. Some of thesesolutions and particularly those containing the enzymes are necessarilyin a concentrated form in order to preserve their activity.

When employing a kit of this type, to assay a specimen, it is necessaryto first reconstitute the components to the required strength by addinga specified amount of another liquid such as water to various solutions.After all of the various components have been reconstituted, theappropriate quantities of each are combined to form the reagent. Apredetermined quantity of the reagent is then mixed with the specimen toproduce the desired assay reactions. The accuracy of the final assay isalso dependent upon the accuracy with which components arereconstituted, the accuracy with which the reconstituted components arecombined to form the resultant reagent and the accuracy with which thereagent is measured when it is mixed with the specimen. It may thus beseen that the accuracy of the assay is dependent upon the skill of theoperator and the accuracy with which he prepares and uses the reagent.

It can be readily appreciated that the foregoing process is verytime-consuming particularly when considering the time for using andcleaning the substantial amounts of equipment such as various pieces ofglassware, measuring instruments, etc. If any of the equipment has anyforeign matter thereon, the reagent may easily be contaminated wherebythe results of the assay will be misleading.

It should also be noted that after the reagent is fully prepared, atleast one of the components therein and particularly the enzymes arequite unstable and rapidly lose their activity. As a consequence, if thereagent is not used within a matter of a few hours following itspreparation, it must be discarded and, therefore, wasted. The percentageof the reagent wasted in this manner becomes very large where only a fewassays are made at infrequent intervals.

It may thus be seen that although the foregoing kits have been capableof producing the desired reactions and permitting the desired assays tobe made they have not been entirely satisfactory for numerous reasons.For example, they have not only been very time-consuming and wasteful,but have also required a person of sufficient skill to insure theaccurate preparation of the reagents and their being used in the propermanner. Also, because of the possibility of substantial human errorssuch reagents have induced a certain degree of unpredictable error inthe results of the assay.

It is an object of the present invention to provide means which will beeffective to overcome the foregoing diificulties. More particularly, itis proposed to provide new and novel assay materials useful in makingbiological assays and the method for preparing the materials. All of theassay materials are in a dry, solid state that may be easily handled andused. The assay materials include components such as enzymes, coenzyrnesand/or substrates which have heretofore been very unstable. Moreover,the combining of such components tends to reduce their stability.However, stabilizers are included that are effective to maintain orpreserve the activity of each of the components and of the entire assaymaterial. Each of the components including those containing the enzymesmay be stabilized individually and used as such for any desired purpose.Also, the compounds may be combined together to form a new and novelassay material. The resultant assay material contains all of thecomponents except Water, for making a liquid reagent that can be used tomake a biological assay of the above described type.

Although the assay material contains various components such as enzymes,coenzyrnes and/or substrates, etc., the material is in a dry, solid formand will be very stable and have a long shelf life. This will permit theassay material to be packaged into containers which are easy to handleand use. Each of the containers may include a quantity of the assaymaterial that is just the right amount for making a particular number ofassays, for example a single assay. The assay material may desirablyinclude an agent which, among other things, is effective to increase thevolume of the assay material to a standard size whereby the quantity ofthe assay material required to produce a single assay will always be astandard amount. In order to make an assay, the contents of one of astandard size container may be mixed with a predetermined quantity ofwater to produce a liquid reagent 3 that is suitable for making a singleassay. In addition this agent can facilitate handling during manufactureand increase shelf life. A preferred agent is mannitol. It will thus beseen that the possibility of human errors is eliminated and a relativelyinexperienced person may prepare the liquid reagent and make the desiredassay without any time-consuming measurements, using any largequantities of glassware, etc.

7 The term enzymatic assay is defined as the use of an t enzyme as acomponent of a reagent for the determination of a substance or anotherenzyme, or for the determination of an enzyme in a biological sample.

These and other objectives and advantages of the present invention willbecome readily apparent from the following detailed description of alimited number of embodiments of the present invention.

The present invention is particularly adapted to be embodied in a dryassay material for determining in a specimen of fluid, particularly ofbiological origin, the quantity or the amount of activity of a certainunknown.

In the present instance, the unknowns are in a class Which forconvenience may be divided into four separate groups. The groups includeenzymes such as carboxylases, dehydrogenases, hydrolases, isomerases,oxidases, phosphorylases and transferases. By way of example, this groupincludes: lactate dehydrogenase, alkaline phosphatase, glucose, oxidase,muscle phosphorylase, glutamateoxaloacetate transaminase,phosphoenolpyruvate carboxylase, cholinesterase, glutamate-pyruvatetransaminase, malate dehydrogenase, acid phosphatase, prostatic acidphosphatase, esterase, diesterase, lipase, amylase, sorbitoldehydrogenase, glucose-6-phosphate dehydrogenase, isocitratedehydrogenase, alpha-hydroxybutyrate dehydrogenase, aldolase, glutamatedecarboxylase, uricase, galactowaldenase, triose phosphate isomerase,carbonic anhydrase, leucine amino-pept'idase, 3-phosphoglyceraldehydedehydrogenase, trypsin and chymotrypsin. Also included are kinases, likecreatine kinase.

The second group includes biochemical intermediates or metabolites. Byway of example, the second group includes: glucose, lactic acid, pynuvicacid, adenosine triphosphate, phenylpyruvic acid, 3methoxy-4-hydroxymandelic acid, cholesterol, creatinine, creatine, urea,uric acid, aspartic acid and glycine.

The third group includes chemical constituents of cells or biologicalfluids which, by way of example, may include dissolved carbon dioxide,triglycerides, protein, starch, glycogen, hemoglobin and insulin.

The fourth group includes drugs and toxins such as antimycin A,diisopropylfluorophosphate, sulfathiazole, ethanol, acetaldehyde andbarbiturates.

To assay a specimen for one of the unknowns within a class, a liquidreagent may be mixed with the specimen to produce an enzymatic reaction.The particular reac tion that occurs should produce an effect which canbe easily measured. By way of example, the optical density of the assaymixture at some predetenmined Wavelength may change in proportion to theextent of the reaction.

In the present instance, the liquid reagent is prepared by dissolving adry, solid assay material in water. When this solution is mixed with thespecimen an assay mixture will be formed that includes a substrate thatwill enter into the reaction, an enzyme that will catalyze the reaction,and a co-enzyme that will be oxidized or reduced in the course of thereaction so as to produce a desired change in the assay mixture, forexample, its optical density. All of the components that are not presentin the specimen are contained in the assay material. In addition, one ormore substances are included in the assay material to stabilize theassay material and preserve the activity of the various components. Inaddition, one or more buifer substances may also be provided that willbe effecttive to maintain the conditions in the assay mixture suitablefor the reaction to occur at an optimum rate.

The assay material is in a dry, powdered form and contains all of thecomponents except water, for producing an enzymatic assay when combinedwith the specimen.

Since the assay material is very stable, the powder may be pre-measuredinto portions which are of just the right amounts for producing an assayreaction in a single specimen or an integral number of specimens. Thepre-measured quantity of the assay material may be dissolved directlyinto a suitable quantity of water to form a liquid reagent. The liquidreagent may then be mixed with the specimen to induce the assay reactionin the assay mixture.

If the unknown being assayed is an enzyme, the reagent will notnecessarily include an enzyme. Accordingly in one embodiment, the assaymaterial is free of any enzymes but includes one or more components suchas a substrate that will react at a rate or to the extent that isdetermined by the amount of activity of the unknnown or enzymeoriginally present in the specimen.

In a second embodiment of the assay material, a subtrate is includedwhich will react with the unknown and an enzyme included in the reagent,that will catalyze the reaction. In order to prepare an assay materialof this embodiment, the first step is to select one or more substratesand one or more enzymes that will be efiective to produce an enzymaticassay reaction and insure its occurring in the desired manner. Theparticular enzymes that are chosen will, of course, depend upon theparticular unknown to be assayed and the particular reaction which it isdesired to create. However, the enzymes will normally be chosen from aclass that includes carboxyl ases, dehydrogenases, hydrolases,isomerases, oxidases, phosphorylases, and transferases. Byway ofexample, this class includes: lactate dehydrogenase, alkalinephosphatase, glucose oxidase, muscle phosphorylase, glutamateoxaloacetate transaminase, phosphoenolpyruvate carboxylase,cholinesterase, glutamate-pyruvate transaminase, malate dehydrogenase,acid phosphatase, prostatic acid phosphatase, esterase, diesterase,lipase, amylase, sorbitol dehydrogenase, glucose-6-phosphatedehydrogenase, isocitrate dehydrogenase, alpha-hydroxybutyratedehydrogenase, aldolase, glutamate, decarboxylase, uricase,galactowaldenase, triose phosphate isomerase, carbonic anhydrase,leucine aminopeptidase, 3-phosphaoglyceraldehyde dehydrogenase, trypsin,chymotrypsin, alpha-hydroxybutyrate dehydrogenase andbeta-hydroxybutyrate dehydrogenase.

As is well known, enzymes out this type are generally very unstable. Infact, heretofore, in order to maintain enzymes of this type in a stablecondition, it has been necessary to keep them in a concentrated form andat low temperatures. In addition to being concentrated, it has also beenusually necessary for the enzyme solution or suspension to include asubstantial amount of a salt such as ammonium sulfate to maintainoptimal activity.

In the present invention, to prepare the assay material in a dry form,one of the steps in the process is to convert the enzyme from a solutionto a dry, solid form such as a powder in which the enzyme is verystable. To accomplish this, one or more stabilizing compounds may beadded to the solution containing the enzymes. The particular stabilizersadded to the enzyme solution will, in part, vary with the particularenzyme that is to be stabilized. However, for enzymes of this type, atleast one stabilizer is chosen from one or more. of the followinggroups. Under some circumstances it has been found to be advantageous toemploy a combination of stabilizers which may include a stabilizer fromseveral of the following groups or even a stabilizer from each group.

Group I. Mucilagenous gums or polysaccharides such gum acacia, gumcaarageenan, tragacanthin, alginic acid and pectin substances. Gumacacia has been found to be particularly well suited for this purpose.In addition to or as an alternative to the gums, the stabilizer mayinclude other polymers containing hydroxy groups or other by .5drophilic substitute groups which render the resultant polymeressentially soluble in water such as polyvinylpyrrolidine, carbowax andpolyvinyl alcohol. This will also insure all of the assay materialdissolving rapidly in the water when the reagent is prepared. However,it is also possible to use any other polymers which because of largechains or hydrophylic substitutent is only partially soluble in waterbut which equilibriate with the aqueous phase such as ion exchangeresins, ion exchange cellulose, carboxymethyl cellulose.

Group II. A buffer consisting of a hydroxyalkylamine including but notlimited to primary amines such as tris (hydroxymethyl) aminomethane or atertiary amine such as triethanolamine.

Group III. A sequestering or complexing agent such as ethylene diaminetetracetic acid or one of its salts which has been found to beparticularly well suited.

Group IV. An inert soluble protein such as bovine serum albumin.

Group V. Salts of a polyvalent anion such as ammonium sulfate, or sodiumpotassium tartrate, which have been found particularly suitable.

Group VI. Sulfhydryl compound such as dithioerythritol, cysteine, orreduced glutathione, which have been found particularly :suitable.

After one or more of the stabilizers of the class described above hasbeen completely dissolved or uniformly dispersed throughout the entiremixture, the enzyme or anzymes in the solution are very stable. It hasbeen found that by adding these stabilizers to the solution, theactivity of the enzymes is often increased. This is believed to resultfrom the elimination of the effects of certain inhibitors which areusually present with the enzymes. In the event it is desired to decreasethe activity of the solution, the solution may be diluted by addingwater. Conversely, if it is desired to concentrate or increase theamount of activity of this enzyme solution, a portion of the liquid maybe removed. Preferably, the liquid is removed by evaporation while thesolution is maintained at a relatively low temperature. It may thus beseen that a very stable enzyme solution is provided at this point andthat the stability of the solution is independent of the concentrationof the enzyme or the salts therein and exhibits stability over a widerrange of temperature.

It is an overall objective to provide an integrated assay material,which is dry, stable, enzymatic, pyridine nucleotide linked, and uniformin results under varying climatic and storage conditions.

The above described stabilized enzyme solution may be used for numerouspurposes as a solution. However, under some circumstances such as thepreparation of the present assay material, it may be desirable toconvert the solution into a dry mixture or powder containing the enzyme.This may be accomplished by lyophilizing or freeze drying the solution.More particularly, the entire solution is frozen to provide a solid massand placed under a vacuum. The vacuum is of sufiicient magnitude tocause the frozen liquid to sublimate. The frozen mass is kept under thisvacuum for a period sufiicient to insure all of the water, etc., beingremoved. This will leave a solid residue that contains the enzyme orenzymes in intimate relation with the stabilizers, such as acacia, etc.These stabilizers are effective in preserving the activity of the enzymefor an extended period of time even though the enzyme is in the form ofa solid.

The term stabilizer, as used herein, broadly relates to a substancewhich prevents the change or destruction of a reagent component. It hasthree major aspects in the present invention, namely: (1) to allow forconvenient handling of the components during manufacture; (2) to permitthe preparation and storage of a component in dry form; and (3) toprovide long-term shelf-life of the finished product.

The residue will normally be in a fluffy or flaky condition. However, ifit is desired, the residue may be ground until it is reduced to a finelypowdered mixture. The grinding may be accomplished by any suitable meanssuch as a ball mill. The fact that the powder mixture is substantiallydry contributes to the chemical stability of the enzymes. It alsocontributes to the physical stability of the mixture and substantiallyeliminates the tendency to compact or become lumpy, etc. Since themixture can be maintained as a loose powder it will be easy to handleand process. Also, it can be easily measured either volumetrically orgravimetrically. At this point a powder is provided that includes one ormore enzymes in a very stable form. Since the powder does not includeany form of substrate, it can be used as an enzyme for any desiredpurpose. For example, among other things, the enzyme powder may beemployed to complete the preparation of the present assay material.

In order to complete the preparation of the assay ma terial the othercomponents such as the buffers, substrates, coenzymes, andbulking-stabilizing agents, may be prepared for mixing with thestabilized enzymes. It is one of the primary purposes of the buffermaterials to maintain the conditions suitable for the assay reaction tooccur at an optimum rate. When the assay material is dissolved in water,the buffers will, among other things, be effective to maintain the pH ofthe liquid reagent. In addition, when the liquid reagent is, in turn,mixed with a specimen containing the unknown, the pH of the resultantspecimen mixture will still be suitable for the assay reaction to occur.

The particular buffer material that is employed in any particular assaymaterial will be dependent upon the particular assay reaction to beconducted and the other components in the assay material. However,normally, they will be in a class that includes the salts of polyvalentinorganic anions and organic amines together with the acids and saltsthereof. By way of example, the salts of polyvalent inorganic anions mayinclude at least sodium and potassium phosphates and sodium andpotassium pyrophosphates. By way of example, organic amines and acids,and their salts may include at least tris (hydroxymethyl) amino-methaneand imidazole (and their salts, such as the hydrochloride, succinate,sulfate), succinic, aspartic, and glutamic acids (and their salts suchas the sodium, potassium, and lithium), glycylglycine, and glycine.

The buffer materials may be prepared in the form of a dry powder that ismixed directly with the lyophilized powder containing the enzyme and thestabilizer. The resultant powder will contain the enzymes required forthe assay reaction. Because of the stabilizers and buffers present inthe powder, the enzymes will be very stable. This resultant powder likethe enzyme powder first described, will not be hygroscopic, in contrastto corresponding mixtures prepared by lyophilization of the combinedbuffer and enzyme solutions.

The substrate is effective for reaction with the unknown in thespecimen. Accordingly, the particular substrate that is employed in anyparticular assay material will be dependent upon the nature of theunknown and the particular assay reaction that it is desired to produce.Normally, the substrate will be in a class of biochemicals whosechemical reactions will be specifically catalyzed by the classes ofenzymes previously described.

The following are specific examples of some of the substrates that fallwithin this class: alanine, alphaor beta-ketoglutaric acid, asparticacid, fructose-1, 6-diphosphate and glucose.

When the assay material is dissolved to form a liquid reagent and thereagent is mixed with the specimen, the substrate will react with theunknown. However, in order for the reaction to occur successfully, it isnecessary for the enzyme to catalyze the reaction. The quantity of thesubstrate and the amount of activity of the enzyme contained in thereagent are in excess of that required to cause all of the unknown tocompletely react or to react at a desired rate. As a result the onlyfactor that limits the assay reaction will be the quantity or amount ofactivity of the unknown.

When the substrates are in a pure solid dry form, they may be groundinto a dry powder suitable for mixing with the lyophilized powder.

The coenzyme enters into the reaction and is converted from one form toanother form. The extent to which the coenzyme is converted isdetermined by the extent to which the assay reaction progresses. Thecoenzyme may be readily converted from one form (such as oxidized) toanother form (such as reduced). In addition the coenzyme has a lightabsorption at some particular wavelength only when it is in one of theseforms. When it is in the other form, it is transparent at the designatedwavelength, although the absorption band may be any desired wavelengththat is convenient to use. However, it is desirable that it be distinctfrom the intense absorption bands of the rest of the components in theassay material and the substances in the specimen. This will insure allof the substances in the reagent and the specimen, except the coenzyme,being transparent or substantially transparent although some of thevarious components may absorb limited quantities of light in the regionof the selected wavelength and they will not vary during the period ofassay whereby the only variable will be the coenzyme in the absorbingform. Thus by measuring the Optical density at the designatedWavelength, the amount of the coenzyme converted may be determined. Morespecifically, by measuring the amount of change or rate of change of theoptical density at the designated wavelength, the amount or rate of theassay reaction may be measured. It has been found that the pyridinenucleotides are particularly well suited for this purpose. When they arein their reduced form, they show absorption of ultraviolet light with amaximum value at a wavelength of about 340 millimicrons. By employing acoenzyme of this class in all forms of the assay material, the assayreactions may be observed by always measuring the optical density atthis Wavelength.

Coenzymes of this class have a limited amount of stability in a solidform. They can be stored in a solid form for only short periods of time.The stability of the coenzyme may be increased by preparing alyophilized powder of the coenzyme and acacia. Further increases in thestability of the coenzyme may be obtained by mixing with mannitol.Accordingly, the coenzyme may be ground into a powder and mixed directlywith the lyophilized powder containing the enzyme, stabilizer, andbuffer.

Heretofore, substrates, enzymes and coenzymes, and buffers have beendissolved in the solution containing the enzyme before lyophilization.The complete solution may then be lyophilized to provide a dry residuecontaining all of the components of the assay. However, it has beenfound, as a practical matter, the resultant assay material is oftenhygroscopic. As a consequence, the residue sometimes tends to absorbvarying or unpredictable quantities of moisture. As a result whenprepared in this manner it is desirable for the resultant mixture to behermetically sealed within a container. However, even when hermeticallysealed, the mixture may still be unstable and tend to develop coloredspecks due to local decomposition and in a fairly short time completelydecompose or lose its activity. It has also been found that the mixturetends to form into lumps which makes it difficult to handle and measureinto small units of identical amounts on a volumetric or weight basis.

This invention teaches that by preparing the various components of theassay material such as the substrates and coenzymes in a dry or solidstate, and in a stabilized form before the mixing thereof with thelyophilized stabilized enzyme, a much more stable and easily handledassay material is now provided.

In addition to the foregoing components, it has also been founddesirable to add a bulking-stabilizing agent to the mixture. This agentmay be a polyhydric substance such as mannitol, sorbitol, lactose,polyvinyl alcohol or polymers having from 1 to 5 hydroxyl groups permonomeric unit. The bulking agent is not active in the assay reaction.Accordingly, the quantity of the bulking agent added to the assaymaterial is not critical and may be varied throughout a wide range.However, the bulking agent performs several unexpected and usefulfunctions. First of all, the :bulking agent tends to further increasethe stability of the assay material for several reasons. Such agentshave the ability to absorb and retain limited quantities of moisturewhereby the assay material is not materially affected when exposed toreasonable amounts of moisture. This increases the stability of theassay material and prevents its losing its activity. It has also beenfound that the bulking agent will also be eflfective in preserving theassay material by increasing the compatibility of its components. It hasalso been found that bulking agents of this category are also effectivein increasing the ability of the assay material to withstand relativelyhigh temperatures, such as 50 C., for longer periods of time.Heretofore, temperatures in this range have caused rapid deteriorationof the enzymes, coenzymes, and other components.

Secondly, it has been found that the use of the bulking agent in theassay material results in the assay material dissolving more rapidlyinto water. This not only reduces the time required for preparing theliquid reagent but also increases the convenience of preparation byreducing the amount of stirring or shaking.

Thirdly, since the bulking agent does not enter into the reaction oraffect the components in the assay material, the quantity of the bulkingagent added to the assay material may vary over a wide range. Once abatch of the assay material has been prepared, its strength or amount ofactivity may be determined. The bulking agent may then be added tostandardize the assay material to a predetermined level. This willresult in the assay material always having a predetermined amount ofactivity per unit irrespective of the batch in which it is prepared. Ofthe agents listed above, mannitol is preferred.

After the assay material has had the bulking agent added, it may bedivided into units of a standard predetermined size. The size normallywill be just large enough to make a single assay or an integral numberof assays. Each of these units may then be packaged into a containersuch as a capsule, glass vial, etc.

It will thus be seen that a plurality of substantially identicalpackages such as foil containers or capsules may be provided. Each ofthese capsules will contain just a sufficient quantity of the assaymaterial for making a single assay of a specimen. In order to make anassay, a package containing the assay material for making the particularassay is selected. The assay material contained in the package is allpre-measured and of a predetermined activity. Accordingly, it may bedissolved directly in a standard amount of water so as to form a liquidreagent. This liquid reagent is then mixed With the specimen to producean enzymatic reaction. The extent of or the rate at which the reactionoccu-rs will be a function of the quantity or amount of activity of theoriginal unknown. Every test, irrespective of the particular type ofassay, will involve the conversion of a coenzyme from one form toanother form wherein one form has an optical absorption at apredetermined wavelength. Accordingly, the optical density of thespecimen at that wavelength will vary as a function of the unknown.Thus, by measuring the optical density of the medium at different times,it will be possible to compute the quantity or amount of activity of theunknown in the original specimen.

The invention sought to be patented in a principal process of makingaspect, is described as residing in the concept of preparing an assayreagent, useful, inter alia, as

an aid in the clinical diagnosis of pathological conditions to determinethe presence of, quantity of, or amount of activity of an enzyme in abiological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent While in substantially anhydrous particulate form,comprising a dry coenzyme, a dry buffer, a dry second enzyme capable ofcatalyzing the conversion of the coenzyme to the other form of thecoenzyme, dry substrates, effective to react with the enzyme of whichthe present quantity or activity is to be determined.

The invention sought to be patented in a second embodiment of aprincipal process of making aspect, is described as residing in theconcept of preparing an assay reagent, useful, inter alia, as an aid inthe clinical diagnosis of pathological conditions to determine thepresence of, quantity of, or amount of activity of an unknown, in abiological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent while in substantially anhydrous particulate form, andcomprising a dry coenzyme, a dry buffer, a dry substrate, effective toreact with the unknown of which the present quantity of activity is tobe determined, a dry first enzyme capable of catalyzing the reaction ofthe unknown with the substrate to form an intermediate product, a drysecond enzyme capable of catalyzing the reaction of the intermediateproduct and the coenzyme.

The invention sought to be patented in a third embodiment of a principalprocess of making aspect, is described as residing in the concept ofpreparing an assay reagent, useful, inter alia, as an aid in theclinical diagnosis of pathological conditions to determine the presenceof, quantity of, or amount of activity of an enzyme in a biologicalspecimen by mixing therewith a lyophilized, stabilized, catalyticreagent While in substantially anhydrous particulate form, comprising adry coenzyme, a dry buffer, and a dry substrate effective to react withthe coenzyme, of which the quantity or activity present is to bedetermined to form the other form of the coenzyme.

The invention sought to be patented in a fourth embodiment of a processof making aspect is described as residing in the concept of stabilizingthe enzyme malate dehydrogenase, by admixing a stabilizer selected fromat least one of tris (hydroxymethyl) aminomethane, its sulfate salt,acacia, ammonium sulfate or ethylenediamine tetraacetic acid.

The invention sought to be patented in a fifth embodiment of a processof making aspect is described as residing in the concept of stabilizingthe enzyme lactate dehydrogenase, by admixing a stabilizer selected fromat least one of tris (hydroxymethyl) aminomethane, its sulfate salt,acacia, ammonium sulfate or ethylenediamine tetraacetic acid.

The invention sought to be patented in a sixth embodiment of a processof making aspect is described as residing in the concept of stabilizingthe enzyme lactate dehydrogenase, by admixing a stabilizer selected fromat least one of polyhydric substances and polymers of said polyhydricsubstances with from 1 to 5 hydroxyl groups per monomeric unit.

The invention sought to be patented in a seventh embodiment of a processof making aspect is described as residing in the concept of stabilizingthe enzyme hexokinase, by admixing a stabilizer selected from at leastone of acacia, tris (hydroxymethyl) aminomethane, its sulfate salt,ammonium sulfate or ethylenediamine tetraacetic acid.

The invention sought to be patented in an eighth embodiment of a processof making aspect is described as residing in the concept of stabilizingthe enzyme triose phosphate isomerase, by admixing a stabilizer selectedfrom at least one of acacia, tris (hydroxymethyl) aminomethane, itssulfate salt, ammonium sulfate or ethylenediamine tetraacetic acid.

The invention sought to be patented in a ninth embodiment of a processof making aspect is described as residing in the concept of stabilizingthe enzyme glyceraldehyde phosphate dehydrogenase, by admixing astabilizer selected from at least one of acacia, tris (hydroxymethyl)aminomethane, its sulfate salt, ammonium sulfate or ethylenediaminetetraacetic acid.

The invention sought to be patented in a tenth embodiment of a processof making aspect is described as residing in the concept of preparing anassay reagent, useful, inter alia, as an aid in the clinical diagnosisof pathological conditions to determine the presence of, quantity of, oramount of activity of glutamate pyruvate transaminase in a biologicalspecimen by mixing therewith a lyophilized, stabilized, catalyticreagent while in substantially anhydrous particulate form including thecombination of the dry substrates alanine and alphaketoglutaric acid,the dry enzyme lactate dehydrogenase, at least one dry stabilizer fromthe class that includes mucilaginous gums, hydroxyalkylamines, ethylenediamine tetraacetic acid and its salts, an inert soluble protein and asulfate anion, a dry coenzyme consisting of reduced diphosphopyridinenucleotide, a dry buffer from the class that includes the salts ofphosphates, organic acids and amines, and the salts of said acids andamines, and at least one dry stabilizing-bulking agent from a class thatincludes polyhydric substances and polymers of said polyhydricsubstances with from 1 to 5 hydroxyl groups per monomeric unit; whichinvolves determining the optical density of the solid reagent afterforming a liquid reagent therefrom and again following a predeterminedincubation period after admixing the specimen.

The invention sought to be patented in an eleventh embodiment of aprocess of making aspect is described as residing in the concept ofpreparing an assay reagent, useful, inter alia, as an aid in theclinical diagnosis of pathological conditions to determine the presenceof, quantity of, or amount of activity of glutamate oxaloacetatetransaminase in a biological specimen by mixing therewith a lyophilized,stabilized, catalytic reagent while in substantially anhydrousparticulate form including the combination of: the dry substrates ofaspartic acid and alpha ketoglutaric acid, the dry enzyme malatedehydrogenase, at least one dry stabilizer from the class that includesmucilaginous gums, hydroxyalkylamines, ethylenediamine tetraacetic acidand its salts, an inert soluble protein and a sulfate anion; a drycoenzyme consisting of reduced diphosphopyridine nucleotide; a bufferfrom the class that includes the salts of phosphates, organic acids andamines, and the salts of said acids and amines; at least one drystabilizing-bulking agent from a class that includes polyhydricsubstances and polymers of said polyhydric sub stances with from 1 to 5hydroxyl groups per monomeric unit; which involves determining theoptical density of the solid reagent after forming a liquid reagenttherefrom, and again following a predetermined incubation period andafter admixing the specimen.

The invention sought to be patented in a twelfth embodiment of a processof making aspect is described as residing in the concept of preparing anassay reagent, useful, inter alia, as an aid in the clinical diagnosisof pathological conditions to determine the presence of, quantity of, oramount of activity of aldolase in a biological specimen by mixingtherewith a lyophilized, stabilized, catalytic reagent while insubstantially anhydrous particulate form including the combination of: adry substrate comprising fructose-1,6-diphosphate; the dry enzymestriose phosphate isomerase and glyceraldehyde phosphate dehydrogenase; adry coenzyme consisting of reduced diphosphopyridine nucleotide; 21 drystabilizer from the class that includes mucilaginous gums,hydroxyalkylamines, ethylene diamine tetracetic acid and its salts, aninert soluble protein and a sulfate anion; a dry buffer from a classthat includes the salts of phosphate, organic acids and amines, and thesalts of said acids and amines; a dry stabilizingbulking agentcomprising polyhydric substances and polymers of said polyhydricsubstances with from 1 to 5 hydroxyl groups per monomeric unit; whichinvolves determining the optical density of the solid reagent afterforming a liquid reagent therefrom, and again following a predeterminedincubation period after admixing the specimen.

The invention sought to be patented in a thirteenth embodiment of aprocess of making aspect is described as residing in the concept ofemploying preparing an assay reagent, useful, inter alia, as an aid inthe clinical diagnosis of pathological conditions to determine thepresence and quantity of glucose in a biological specimen by mixingtherewith a lyophilized, stabilized, catalytic reagent while insubstantially anhydrous particulate form, including the combination of:the dry enzymes hexokinase and glucose-6-phosphate dehydrogenase; a drycoenzyme consisting of diphosphopyridine nucleotide; a dry stabilizerfrom the class that includes mucilageous gums, hydroxyalkylamines,ethylenediamine tetraacetic acid and its salts, an inert solubleprotein, and a sulfate anion; a dry buffer from the class that includesmucilaginous gums, hydroxyorganic acids and amines, and the salts ofsaid acids and amines; a dry stabilizing-bulking agent comprisingpolyhydric substances with from 1 to 5 hydroxy groups per monomericunit; Which involves determining the optical density of the solidreagent after forming a liquid reagent therefrom, and again afteradmixing the specimen and allowing sufi'icient time for the mixture toreact completely.

The invention sought to be patented in a fourteenth embodiment of aprocess of making aspect is described as residing in the concept ofpreparing an assay reagent, useful, inter alia, as an aid in theclinical diagnosis of pathological conditions to determine the presenceof, quantity of, or amount of activity of adenosine triphosphate in abiological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent while in substantially anhydrous particulate formincluding the combination of: the dry enzymes hexokinase andglucose-6-phosphate dehydrogenase; a substrate comprising glucose; a drycoenzyme consisting of triphosphopyridine nucleoxide; a dry stabilizerfrom the class that includes mucilaginous gums, hydroxyalkylamines,ethylene diamine tetraacetic acid and its salts, an inert solubleprotein and a sulfate anion; a dry butfer from a class that includes thesalts of phosphate anions, organic acids and amines, and the salts ofsaid acids and amines, a dry stabilizing-bulking agent comprisingpolyhydric substances and polymers of said polyhydric substances withfrom 1 to 5 hydroxyl groups per monomeric unit; which involvesdetermining the optical density of the solid reagent after forming aliquid reagent therefrom, and again after admixing the specimen andallowing sufficient time for the mixture to react completely.

The invention sought to be patented in a fifteenth embodiment of aprocess of making aspect is described as residing in the concept ofpreparing an assay reagent, useful, inter alia, as an aid in theclinical diagnosis of pathological conditions to determine the presenceof, quantity of, or amount of activity of malate dehydrogenase in abiological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent while in substantially anhydrous particulate formincluding the combination of: the dry substrate comprising oxaloaceticacid; the dry coenzyme reduced diphosphopyridine nucleotide; a drystabilizingbulking agent from the class that includes polyhydricsubstances and polymers of said polyhydric substances with from 1 to 5hydroxyl groups per monomeric unit; and a dry buffer from the class thatincludes the salts of phosphate anions, organic acids and amines, andthe salts of said acids and amines; which involves determining theoptical density of the solid reagent after forming a liquid reagenttherefrom, and again following a predetermined incubation period afteradmixing the specimen.

The invention sought to be patented in a sixteenth embodiment of aprocess of making aspect is described as residing in the concept ofpreparing an essay reagent,

useful, inter alia, as an aid in the clinical diagnosis of, pathologicalconditions to determine the presence of, quantity of, or amount ofactivity of lactate dehydrogenase in a biological specimen by mixingtherewith a lyophilized, stabilized, catalytic reagent while insubstantially anhydrous particulate form including the combination of:the dry substrate comprising sodium pyruvate; a dry coenzyme reduceddiphosphopyridine nucleotide; a dry stabilizing-bulking agent from theclass that includes polyhydric substances with from 1 to 5 hydroxylgroups per monomeric unit; and a dry buffer from the class that includesthe salts of phosphate anions, organic acids and amines, and the saltsof said acids and amines; which involves determining the optical densityof the solid reagent after forming a liquid reagent therefrom, and againfollowing a predetermined incubation period after admixing the specimen.

The invention sought to be patented in a seventeenth embodiment of aprocess of making aspect is described as residing in the concept ofpreparing an assay reagent, useful, inter alia, as an aid in theclinical diagnosis of pathological conditions to determine the presenceof, quantity of, or amount of activity of alpha-hydroxybutyratedehydrogenase in a biological specimen by mixing therewith a lypholized,stabilized, catalytic reagent while in substantially anhydrousparticulate form including the combination of: the dry substrate sodiumalphaketobutyrate; a coenzyme reduced diphosphopyridine nucleotide; adry stabilizing-bulking agent from the class that includes polyhydricsubstances and polymers of said polyhydric substances with from 1 to 5hydroxyl groups per monomeric unit; and a dry buffer consisting of thealkali metal phosphates or tris (hydroxymethyl) aminomethane andsuccinic acid; which involves determining the optical density of thesolid reagent after forming a liquid reagent therefrom, and againfollowing a predetermined incubation period after admixing the specimen.

The invention sought to be patented in an eighteenth embodiment of aprocess of making aspect is described as residing in the concept ofpreparing an assay reagent, useful, interalia, as an aid in the clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of glucose-6-phosphate dehydrogenasein a biological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent While in substantially anhydrous particulate formincluding the combination of: the dry substrates sodium salt of glucose6 phosphate; a coenzyme triphosphopyridinenucleotide; a drystabilizing-bulking agent from the class that includes polyhydricsubstances and polymers of said polyhydric substances with from 1 to 5hydroxyl groups per monomeric unit; and a dry buffer from the class thatincludes alkali metal phosphates or tris (hydroxymethyl) aminomethaneand succinic acid; which involves determining the optical density of thesolid reagent after forming a liquid reagent therefrom, and againfollowing a predetermined incubation period after admixing the specimen.

The invention sought to be patented in a nineteenth embodiment of aprocess of making aspect is described as residing in the concept ofpreparing an assay reagent, useful, inter alia, as an aid in theclinical diagnosis of pathological conditions to determine the presenceand quantity of urea in a biological specimen by mixing therewith alyophilized, stabilized, catalytic reagent while in substantiallyanhydrous particulate form including the combination of: a dry substratecomprising alphaketoglutaric acid; the dry enzymes comprising urease andglutamic dehydrogenase; a dry coenzyme from a class that includes thepyridine nucleotides; preferably a dry activator; a dry stabilizercomprising dithioerythritol Y and sodium potassium tartrate; and a drybutfer that includes at least one of sodium or potassium phosphate, tris(hydroxymethyl) aminomethane, sodium or potassium bicarbonate, sodium orpotassium carbonate and glycine; which involves determining the opticaldensity of the solid reagent after forming a liquid reagent therefrom,and again after admixing the specimen and allowing sufiicient time forthe mixture to react completely.

The invention sought to be patented in a twentieth embodiment of aprocess of making aspect is described as residing in the concept ofpreparing an assay reagent, useful, inter alia, as an aid in theclinical diagnosis of pathological conditions to determine the presenceof, quantity of, or amount of activity of creatine phosphokinase in abiological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent while in substantially anhydrous particulate formincluding the combination of: a dry substrate comprising creatinephosphate; the dry enzymes hexokinase and glucose-6-phosphatedehydrogenase; a dry coenzyme comprising adenosine triphosphate andtriphosphopyridine nucleotide; a dry buffer capable of maintaining thepH between 6.5 and 7.5; at least one dry stabilizer selected frommucilaginous gums, hydroxyalkylamines, ethylenediamine tetraacetic acidand its salts, an inert soluble protein and a sulfate anion; andpreferably dry activator; which involves determining the optical densityof the solid reagent after forming a liquid reagent therefrom, and againfollowing a predetermined incubation period after admixing the specimen.

The invention sought to be patented in a twenty-first embodiment of aprocess of making aspect is described as residing in the concept ofpreparing an assay reagent, useful, inter alia, as an aid in theclinical diagnosis of pathological conditions to determine the presenceof, quantity of, or amount of activity of lactate dehydrogenase in abiological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent while in substantially anhydrous particulate formincluding the combination of: the dry substrate comprising lithiumlactate; a dry coenzyme diphosphopyridine nucleotide; a drystabilizing-bulking agent comprising polyhydric substances with from 1to 5 hydroxyl groups per monomeric unit; and a dry buffer from the classthat includes the alkali metal phosphates and glycine-rhodium carbonatewhich involves determining the optical density of the solid reagentafter forming a liquid reagent therefrom, and again following apredetermined incubation period after admixing the specimen.

The invention sought to be patented in a first composition of matteraspect, is described as residing in the concept of a substantiallyanhydrous solid reagent, useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of an enzyme in a biological specimenby mixing therewith a lyophilized, stabilized, catalytic reagent whilein substantially anhydrous particulate form and comprising a buttercapable of maintaining a predetermined pH of a mixture of the assayreagent and specimen being assayed; substrate(s) effective to react withthe enzyme of which the present quantity of activity is to to bedetermined to form an intermediate product, a coenzyme capable ofconversion from one form to another during use, and a second enzymecapable of catalyzing the reaction of the intermediate product(s) withthe coenzyme, to form the other form of the coenzyme.

The invention sought to be patented in a second composition of matteraspect, is described as residing in the concept of a substantiallyanhydrous solid reagent, useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of an unknown substance in abiological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent while in substantially anhydrous particulate form andcomprising a buffer capable of maintaining a predetermined pH of amixture of the assay reagent and specimen being assayed; a substrateeffective to react with the unknown substance of which the presentquantity or activity is to be determined; an

enzyme capable of catalyzing the reaction of the substance with thesubstrate to form one form to another during use; and, a second enzymecapable of catalyzing the reaction of the intermediate product and thecoenzyme to form the other form of the coenzyme.

The invention sought to be patented in a third composition of matteraspect, is described as residing in the concept of a substantiallyanhydrous solid reagent, useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of an enzyme in a biological specimenby mixing therewith a lyophilized, stabilized, catalytic reagent whilein substantially anhydrous particulate form, and including a buffercapable of maintaining a predetermined pH of a mixture of the assayreagent and specimen being assayed, a coenzyme capable of conversionfrom one form to another during use, a substrate effective to react withthe coenzyme catalyzed by the enzyme, of which the present quantity oractivity is to be determined to form the other form of the coenzyme.

The invention sought to be patented in a fourth composition of matteraspect, is described as residing in the concept of a substantiallyanhydrous solid reagent, useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of glutamate pyruvate transaminase ina biological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent while in substantially anhydrous particulate formincluding the combination of: the dry substrates of alanine andalpha-ketoglutaric acid, the dry enzyme lactate dehydrogenase, at leastone dry stabilizer from the class that includes mucilaginous gums,hydroxyalkylamines, ethylene diamine tetraacetic acid and its salts, aninert soluble protein and a sulfate anion, a dry coenzyme consisting ofreduced diphosphopyridine nucleotide, a dry buffer from the class thatincludes the salts of phosphates, organic acids and amines, and thesalts of said acids and amines, and at least one dry stabilizing-bulkingagent from a class that includes polyhydric substances with from 1 to 5hydroxyl groups per monomeric unit.

Each substance above is present in that quantity so as to insure auniform rate of reaction catalyzed by the unknown being determined, orcause the reaction to go to completion. The above requirement applies toeach of the following described composition aspects of the invention.

The invention sought to be patented in a fifth composition of matteraspect is described as residing in the concept of a substantiallyanhydrous solid reagent, useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of glutamate oxaloacetatetransaminase in a biological specimen by mixing therewith a lyophilized,stabilized, catalytic reagent while in a substantially anhydrousparticulate form including the combination of: the dry substrates ofaspartic acid and alpha ketoglutaric acid; the dry enzyme malatedehydrogenase; at least one dry stabilizer from the class that includesmucilaginous gums, hydr-oxyalkylamines, ethylenediamine tetraacetic acidand its salts, an inert soluble protein and a sulfate anion; a drycoenzyme consisting of reduced diphosphopyridine nucleotide; a drybufier from the class that includes the salts of phosphates, organicacids and amines, and the salts of said acids and amines; at least onedry stabilizing-bulking agent from a class that includes polyhydricsubstances and polymers of said polyhydric substances with from 1 to 5hydroxyl groups per monomeric unit.

The invention sought to be patented in a sixth composition of matteraspect is described as residing in the concept of a substantiallyanhydrous solid reagent, useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of aldolase, in a biological specimenby mixing therewith a lyophilized, stabilized, catalytic reagent whilein substantially anhydrous particulate form including the combinationof: a dry substrate comprising fructose-1,6-diphosphate; the dry enzymestriose phosphate isomerase and glyceraldehyde phosphate dehydrogenase; adry coenzyme consisting of diphosphopyridine nucleotide; a drystabilizer from the class that includes mucilaginous gums,hydroxyalkylamines, ethylene diamine tetraacetic acid and its salts, aninert soluble protein and a sulfate anion; a dry buffer from a classthat includes the salts of phosphate, organic acids and amines, and thesalts of said acids and amines, a dry stabilizing-bulking agentcomprising polyhydric substances and polymers of said polyhydricsubstances with from 1 to 5 hydroxyl groups per monomeric unit,

The invention sought to be patented in a seventh composition of matteraspect is described as residing in the concept of a substantiallyanhydrous solid reagent, useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence andquantity of glucose, in a biological specimen by mixing therewith alyophilized, stabilized, catalytic reagent while in substantiallyanhydrous particulate form including the combination of: the enzymeshexokinase and glucose-6- phosphate dehydrogenase; the dry coenzymesconsisting of triphosphopyridine nucleotide and adenosine triphosphate;a dry stabilizer from the class that includes mucilag inous gums,hydroxyalkylamines, ethylenediamine tetraacetic acid and its salts, aninter soluble protein and a sulfate anion; a dry buffer from a classthat includes the salts of phosphate anions, organic acids and amines,and the salts of said acids and amines; and a dry stabilizingbulkingagent comprising polyhydric substances and polymers of said polyhydricsubstances with from 1 to 5 bydroxyl groups per monomeric unit.

The invention sought to be patented in an eighth composition of matteraspect is described as residing in the concept of a substantiallyanhydrous solid reagent, useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of adenosine triphosphate, in abiological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent while in substantially anhydrous particulate formincluding the combinations of: the dry enzymes hexokinase andglucose-6-phosphate dehydrogenase; a dry substrate comprising glucose; adry coenzyme consisting of triphosphopyridine nucleotide; a drystabilizer from the class that includes mucilagenous gums,hydroxyalkylamines, ethylene diamine tetracetic acid and its salts, aninert soluble protein and a sulfate anion; a dry buffer from a classthat includes the salts of phosphate anions, organic acids and amines,and the salts of said acids and amines; a dry stabilizing-bulking agentcomprising polyhydric substances and polymers of said polyhydricsubstances with from 1 to 5 hydroxy groups per monomeric unit.

The invention sought to be patented in a ninth composition of matteraspect is described as residing in the concept of a substantiallyanhydrous solid reagent, useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of malate dehydrogenase in abiological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent While in substantially anhydrous particulate formincluding the combination of: the dry substrate comprising oxaloaceticacid; the dry coenzyme reduced diphosphopyridine nucleotide; a drystabilizing-bulking agent from the class that includes polyhydricsubstances and polymers of said polyhydric substances with from 1 to 5hydroxyl groups per monomeric unit; and a dry buifer from the class thatincludes the salts of phosphate anions, organic acids and amines, andthe salts of said acids and amines.

The invention sought to be patented in a tenth composition of matteraspect is described as residing in the concept of a substantiallyanhydrous solid reagent, useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of lactate dehydrogenase in abiological specimen by mixing therewith ta lyophilized, stabilized,catalytic reagent while in substantially anhydrous particulate formincluding the combination of: the dry sub strate comprising sodiumpyruvate; the dry coenzyme reduced diphosphopyridine nucleotide; a drystabilizingbulking agent from the class that includes polyhydricsubstances with from 1 to 5 hydroxyl groups per monomeric unit; and adry bufler from the class that includes the salts of phosphate anions,organic acids and amines, and the salts of said acids and amines.

The invention sought to be patented in an eleventh composition of matteraspect is described [as residing in the concept of a substantiallyanhydrous solid reagent useful, inter alia, as an aid in clinicialdiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of alpha-hydroxybutyratedehydrogenase in a biological specimen by mixing therewith alyophilized, stabilized, catalytic reagent While in substantiallyanhydrous particulate form including the combination of: the drysubstrate sodium alphaketobutyrate; the dry coenzyrne reduceddiphosphopyridine nucleotide; a dry stabilizing bulking agent from theclass that includes polyhydric substances and polymers of saidpolyhydric substances with from 1 to 5 hydroxyl groups per monomericunit; and a dry buffer consisting of the alkali metal phosphates or tris(hydroxymet-hyl) aminomethane and succinic acid.

The invention sought to be patented in a twelfth composition of matteraspect is described as residing in the concept of a substantiallyanhydrous solid reagent useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of glucose-6-phosphate dehydrogenasein a biological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent while in substantially anhydrous particulate formincluding the combination of: the dry substrate sodium salt ofglucose-6- phosphate; the dry coenzyme triphosphopyridine nucleotide; adry stabilizing-bulking agent from the class that includes polyhydricsubstances and polymers of said polyhydric substances with from 1 to 5hydroxyl groups per monomeric unit; and a dry buffer from the class thatincludes alkali metal phosphates or tris(hydroxymethy1) aminomethane andsuccinic acid.

The invention S ught to be patented in a thirteenth composition ofmatter aspect is described as residing in the concept of a substantiallyanhydrous solid reagent useful, interalia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of urea in a biological specimen bymixing therewith a lyophilized, stabilized, catalytic reagent while insubstantially anhydrous particulate form including the combination of:the dry substrate alphaketoglutaric acid; the dry enzymes urease andglutamic dehydrogenase; a dry coenzyme from: a class that includes thepyridine nucleotides; a dry stabilizer comprising dithioerythritol andsodium potassium tartrate; and a dry buifer that includes at least oneof sodium or potassium phosphate, tris-(hydroxymethyl) aminomethane,sodium or potassium bicarbonate, sodium or potassium carbonate andglycine, and preferably, a dry activator.

The invention sought to be patented in a fourteenth composition ofmatter aspect is described as residing in the concept of a substantiallyanhydrous solid reagent useful, interalia, as an aid in clinicaldiagnosis of pat-hological conditions to determine the presence of,quantity of, or amount of activity of creatine phosphokinase in abiological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent while in substantially anhydrous particulate formincluding the combination of:

a dry substrate creatine phosphate; the dry enzymes hexokinase andglucoseb-phosphate dehydrogenase; and dry coenzymes adenosinetriphosphate and triphosphopyridinenucleotide; a dry buffer capable ofmaintaining the pH between 6.5 and 7.6; at least one dry stabilizerselected from mucilagenous gums, hydroxyalkylamines, ethylenediaminetetracetic acid and its salts, an inert soluble protein and a sulfateanion; and preferably a dry activator.

The invention sought to be patened in a fifteenth composition of matteraspect is described as residing in the concept of a substantiallyanhydrous solid reagent useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of lactate dehydrogenase in abiological specimen by mixing therewith a lyophilized, stabilized,catalytic reagent while in substantially anhydrous particulate formincluding the combination of: the dry substrate lithium lactate; the drycoenzyme diphosphopyridine nucleotide; a dry stabilizing-bulking agentcomprising polyhydric substances with from 1 to hydroxyl groups permonomeric unit; and a dry buffer from the class that includes the alkalimetal phosphates and glycine-sodium carbonate.

The invention sought to be patented in a sixteenth composition of matteraspect is described as residing in the concept of a substantiallyanhydrous solid reagent useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of a substance in a biologicalspecimen by mixing therewith a lyophilized, stabilized, catalyticreagent while in substantially anhydrous particulate form including thecombination of: hexokinaseand at least one dry stabilizer from the classconsisting of acacia, ammonium sulfate, tris(hydroxymethyl)aminomethane, and its sulfate salt.

The invention sought to be patented in a seventeenth composition ofmatter aspect is described as residing in the concept of a substantiallyanhydrous solid reagent useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of a substance in a biologicalspecimen by mixing therewith a lyophilized, stabilized, catalyticreagent while in substantially anhydrous particulate form including thecombination of: triose phosphate isomerase and a stabilizer thatincludes one of ammonium sulfate and acacia.

The invention sought to be patented in a eighteenth composition ofmatter aspect is described as residing in the concept of a substantiallyanhydrous solid reagent useful, inter alia, as an aid in clinicaldiagnosis of pathological conditions to determine the presence of,quantity of, or amount of activity of an unknown in a biologicalspecimen by mixing therewith a lyophilized, stabilized, catalyticreagent while in substantially anhydrous particlate form including thecombination of: glyceraldehyde phosphate dehydrogenase and a stabilizerthat includes one or more sulfate and acacia.

EXAMPLE A The first example of the first embodiment is a solid reagentor assay material that is particularly adapted to be employed formeasuring the amount of activity of glutamatepyruvate transaminase (GPT)present in a serum. This reagent when fully prepared will consist of thedry mixture of the following substances:

Enzyme: lactate dehydrogenase (LDH) Buffer: phosphate buffer (NaH PO +NaHPO Stabilizer: tris-(hydroxymethyl)-aminomethane and its sulfate salt,ammonium sulfate, ethylenediamine tetraacetic acid, acacia and albuminSubstrate: alanine and alpha-ketoglutaric acid Coenzyrne: r e d u c e ddiphosphopyridine nucleotide (DPNH) Bulking agent: mannitol In order toprepare a large number of units of this assay material or reagent, thefollowing procedure may be employed to produce a batch of a dry assaymaterial or reagent that may then be divided into small quantities andpackaged in containers such as capsules. Wherever quantities arespecified, they are suitable for preparing a batch that will yield about10,000 capsules. However, it should be understood that these values maybe varied if it is desired to produce larger or smaller batches.

The first step in the procedure is to prepare a tris- EDTA stabilizersolution. This may be accomplished by mixing the chemicals together inapproximately the indicated quantities;

Tris (hydroxymethyl) aminomethane 5001,000

Ammonium sulfate 250-500 Ethylenediamine tetraacetic acid,

tetrasodium salt -300 In order to mix these chemicals together, the tris(hydroxymethyl) aminomethane is dissolved in a volume of water that issomewhat less than 1 liter to form a first solution. If it is necessaryto assist in this dissolving, the solution may be heated in a waterbath. After the tris is completely dissolved, the solution is allowed tocool to about 25 C. and adjusted to about pH 7.5. This adjustment may beaccomplished by adding sulfuric acid in the necessary quantities toconvert part of the tris (hydroxymethyl) aminomethane to its sulfatesalt. After the solution is brought to pH 7.5, sufficient water is addedto bring the total volume to 1 liter.

Following this, a second solution is prepared by dissolving the ammoniumsulfate in 850 to 900 ml. of water. This solution is adjusted to pH 7.5by the addition of ammonium hydroxide. The first and second solutionsmay then be mixed with each other to form a resultant solution. Theethylenediamine tetraacetic acid, tetrasodium salt, may then be added tothe resultant solution and dissolved therein to form a bulfer solutionwhich is sometimes hereinafter referred to as the tris-EDTA buffer.

A dry, lyophilized powder containing the lactate dehydrogenase (LDH)enzyme is then prepared. However, before this powder can be prepared, itis necessary to determine first the amount of activity in the lactatedehydrogenase to be added.

In order to insure that the capsules produced from successive batcheswill be of identical activity, the activity of the LDH should bestandardized at a predetermined level. By way of example, in a batch of10,000 capsules, there may be about 20 10 Wroblewski units [Wroblewski,F., and LaDue, T. 8., Proc. Soc. Exp. Biol. Med. 90, 210 (1955)] for theentire batch, or about 2,000 units per finished capsule. The assay maybe made by employing the following chemicals in about the indicatedamounts:

Phosphate buffer, 0.1 M, pH 7.5 1.75 Sodium pyruvate (3.3 mg./ 10 ml. ofphosphate buffer: reagent 1) 1.00

DPNH solution, 2.5 mg./ml. of 1% NaHCO soln 0.15

To begin the assay, the enzyme lactate dehydrogenase is diluted one partto 10,000 by mixing it with the phos phate buffer. A small quantity ofthe diluted enzyme, such as 0.1 m1. is mix-ed into a suitable quantityof the buffer solution containing the pyruvate and DPNH. The opticaldensity of the mixture at a constant temperature such as 32 C. is thenmeasured at suitable intervals such as one minute for an extended periodsuch as ten minutes. By determining the average rate of change perminute of the optical density and multiplying by ten (if 0.1 ml. isused), the units of enzyme per milliliter of the diluted enzyme can bedetermined. Then, dividing by the number of milligrams of enzyme permilliliter, it will be possible to find the number of units of enzymeper milligram.

Lactate dehydrogenase (the exact amount is determined by the aboveassay) mg 90l00 Acacia m arabic, white powder, U.S.P.) g 2-6 Albumin mg50-500 Tris-EDTA solution ml 10-30 Ammonium sulfate g l5-40 Thesechemicals may be mixed as follows. First, dissolve the acacia in aconvenient quantity of distilled water, for example 80 to 160milliliters. The albumin is then dissolved in a second quantity of water(for example, milliliters). When fully dissolved, the two solutions aremixed. The tris-EDTA prepared in the beginning is then added to thesolution. The solution is now placed under a vacuum for a period of timethat will assure all of the trapped air in the solution being removed.Th amount of the enzyme lactate dehydrogenase determined by the assay isthen added to the solution. The resultant solution is then completelymixed to insure a uniform dispersion of the enzyme throughout the entiresolution. The solution is then frozen and a vacuum applied to remove allof the water. The resultant lyophilized mixture will now be in a drysolid state in a quantity in the general range of about to grams. Thisdry mixture may then be mixed with the dry crystals of ammonium sulfate.The dry mixture of solids may then be pulverized into a fine powder byany suitable means such as placing it in a ball mill for several hours.

After the powder is completely mixed and pulverized to a sufficientdegree, an assay may be made of the mixture to determine the units ofactivity of the enzyme lactate dehydrogenase in each milligram of thelyophilized powder. The next step is to distribute the lyophilizedpowder into the capsules. However, if it is desired to delay this step,the lyophilized powder may be stored for extended periods of time in acold place such as a refrigerator or freezer. If this is done, it isdesirable to include a drying agent near the powder to prevent theabsorption of any moisture.

In order to make the capsules, the following are mix d together.Normally, the quantities will be in about the indicated ranges:

Sodium carbonate (to give pH 7.4 to

7.6) g 10 Sodium phosphate, dibasic g 3501,050 D,L-alanine g 1,250-*-10% Alpha-ketoglutaric acid g 60:10% DPNH g 6 to 8 Lactatedehydrogenase, lyophilized units 20 l0 The first three compounds in theabove list are first ground together to form a dry mixture. It has beenfound preferable to use D,L-alanine recrystallized from water only.L-alanine may be substituted for D,L-alanine, in which case, the amountused is one half that for the D,L compound. The mixture is then placedunder a vacuum at a temperature of about 50 C. for a sufficient numberof hours to insure all of the moisture being r moved. In addition, adrying material such as phosphorous pentoxide may be included in theoven to insure a complete drying. After the foregoing drying step, thealpha-ketoglutaric acid is then added to the mixture formed by the firstthree compounds. The alanine and alpha-ketoglutaric acid form asubstrate for the reaction that takes place when assaying the serum forGPT. Accordingly, the exact amounts of these compounds to be used aredetermined by the optimum amounts needed to produce a satisfactoryreaction. Normally, these will be within the range indicated in theabove table.

The resultant mixture of the four compounds is then 20 pulverized into afine powder, for example, by placing in a ball mill for several hours.When completely mixed into a homogeneous mixture, a small sample may bedissolved in water and the pH determined. If it is necessary, the pHshould be adjusted so as to fall within the range of 7.4 to 7.6. If thepH is too low, additional sodium carbonate is added. If the pH is toohigh, a new mixtur similar to the above is prepared in the same manner.However, this mixture is made definitely deficient in sodium carbonate.The second mixture is then blended with the first mixture to bring thepH into the range of 7.4 to 7.6. When the mixture is finished, it isdried again as d scribed above. From this point forward it is importantthat the powder not be exposed to moisture. Generally, it is best thatif the powder is to be exposed to the atmosphere, the relative humidityshould be below 15%.

The pulverized DPNH and the lyophilized powder containing the lactatedehydrogenase may then be added to the powder mixture just preparedabove. Before the DPNH is added, it is dried under vacuum in thepresence of phosphorous pentoxide to remove moisture. At the time thatthe lyophilized powder is prepared, the amount of activity of thelactate dehydrogenase is assayed and a quantity of LDH is computed thatwould produce a total of 20x10 Wroblewski units for the entire batch (if10,000 capsules are to be prepared), or 2,000 units per capsule orsingle assay portion. Accordingly, the quantity of the LDH to be blendedinto the mixture will be determined from that assay. The quantity ofDPNH to be employed is chosen to produce on optical density that iscompatible with spectrometers that are suitable for measuring theoptical density during the reaction. Normally, the quantity of DPNH willbe such as to produce an optical density of the order of 0.8 at 340millimicrons. This may 'be checked by dissolving an appropriate quantityof the powder in water (for example, about 4 mg. per 5 ml.) andmeasuring the optical density.

At this point, a powder is provided that contains the enzyme lactatedehydrogenase, the coenzyme DPNH, and all of the buffers, substrates andstabilizers, to insure the powder being in a stable state that will havea very long shelf life. A sample of this powder is withdrawn (beingcareful not to introduce moisture) and tested. The tests are for theappropriate optical density, homogeneity with respect to the DPNH, andthe elfectiveness of the powder to form an assay for glutamate-pyruvatetransaminase. Accordingly, this powder may be divided into a pluralityof small parts that are just large enough to contain the desired 2,000Wroblewski units and appropriate quantities of buffer, DPNH, L-alanineand alpha-ketoglutarate to perform a GPT determination in 3 ml. Eachpart may then be enclosed in a suitable package such as a capsule thatwill protect the mixture from any moisture.

If a pharmaceutical gelatin capsule is used, a desiccant such as analumina pellet should be placed with the capsule within the package.

Normally, between successive batches, there will be some variations inthe size of the parts into which the mixture is divided as a result ofvariations in the activity of the LDH. In order to make all of the partsof identical size in all batches, a standard package size in excess ofthe largest size that will be required is chosen. A suitable quantity ofa bulking and stabilizing agent such as mannitol may then be added tothe mixture to bring its volume up to an amount that will insure each ofthe parts having a volume that will just fill the package.

In order to make an assay of a serum for glutamatepyruvate transaminaseor GPT using a capsule of this example, a specimen of the serum is firstobtained in a qgantity such as 0.1 milliliter. Following this, thecontents of a capsule of this type are dissolved. The resultant solutionwill form a liquid reagent that is of just the right size to make asingle assay and may be mixed directly with the specimen. As soon as thereagent and specimen serum are mixed together, the following reactionswill occur:

GPT alpha-ketoglutarate alanine glutamic acid pyruvate pyruvate DPNHlactate DPN Since the alpha-ketoglutarate, alanine and DPNH are suppliedin the capsule in excess of that required for the reactions, the onlylimiting factor is the activity of GPT. The pyruvate formed will thus beproportional to the GPT. When the pyruvate reacts with DPNH, it will beconverted to DPN in direct proportion of the pyruvate and, therefore, tothe GPT. By placing the assay mixture in a suitable spectrophotometerand measuring the optical density at 340 millimicrons, the rate at whichDPNH is converted can be measured. This, in turn, will permit the amountof GPT originally present to be determined.

EXAMPLE B The second example of the first embodiment is a solid reagentthat is particularly adapted to be employed for measuring the amount ofactivity of glutamate-oxaloacetic transaminase, or GOT, present in theserum. This reagent when fully prepared will consist of a dry mixturecontaining the following substances:

Enzyme-malate dehydrogenase (MDH) Buffer-disodium hydrogen phosphateStabilizer-acacia, and tris (hydroxymethyl)-amino methane and itssulfate salt, ammonium sulfate and ethylenediamine tetraacetic acidSubstrateaspartic acid and alpha-ketoglutaric acid C-oenzymereduceddiphosphopyridine nucleotide (DPNH) Bulking agent-mannitol In order toprepare a large number of units of this reagent, the following proceduremay be employed to produce a batch of dry reagent that may then bedivided into a large number of small quantities and packaged intocapsules. Wherever quantities for a substance are specified, thequantities are suitable for preparing a batch of reagent that will yieldabout 10,000 capsules. It should be understood, however, that thesevalues are for illustrative purposes only and may be varied to satisfythe demands of the particular application to which the reagent is to beused.

The first step in preparing such a batch is normally to prepare atris-EDTA stabilizer. This buffer is a mixture of tris (hydroxymethyl)aminomethane and its sulfate salt, ammonium sulfate, and ethylenediaminetetraacetic acid, tetrasodium salt, and may be prepared in the samemanner described in Example A.

Following this, a dry, lyophilized powder containing the malatedehydrogenase or MDH is prepared. This may be accomplished by employingthe following chemicals in the indicated ranges:

Malate dehydrogenase units 3-6 10 Gum acacia gm 2-10 Tris-EDTA buffer ml1-4 The acacia may first be dissolved in a suitable quantity of watersuch as about 50 milliliters. This solution may then be mixed with themalate dehydrogenase solution and the tris-EDTA solution. After thesolution has been thoroughly mixed, it may be placed under a vacuum fora sufiicient interval of time to remove any entrapped air. Followingthis, the solution may be frozen and placed under a vacuum until all ofthe moisture is removed therefrom. This will produce a dry lyophilizedpowder which contains the enzyme malate dehydrogenase together withstabilizers which will be effective to maintain the activity of themalate dehydrogenase at its desired level.

After the lyophilized powder containing the malate dehydrogenase hasbeen prepared, it may be assayed to determine the amount of activity ofenzyme malate dehydrogenase present in the powder. This may beaccomplished by employing a reagent containing the following chemicalsin the indicated quantities:

M1. Oxaloacetic acid, 1 mgjml. 0.1 DPNH, 2 mg./ml. 0.1 Phosphate buffer,0.1 M, pH 7.5 2.7

After the foregoing solutions have been prepared in the indicatedconcentrations, they may be all mixed together to form a liquid reagentsuitable for performing the present assay. A suitable quantity of thelyophilized powder containing the enzyme malate dehydrogenase may bedissolved in water. This may be accomplished by dissolving about 20milligrams of powder in about 20 milliliters of water. About 0.02milliliter of this solution may then be combined with the reagent. Whenthis mixture is made, the DPNH will immediately begin to be convertedinto DPN. While this reaction is occurring, the solution may be placedin a suitable spectrophotometer and the optical density in the reagentmeasured at suitable increments of time, for example, every minute.After several readings have been taken, an average may be provided whichwill indicate the amount of activity of the malate dehydrogenase in thelyophilized powder. A change in the optical density of 0.001 per minuteindicates an activity of 1 unit. From this, it will be possible tocalculate the amount of the lyophilized powder containing the malatedehydrogenase that is required to produce an activity in the ranges ofabout 8 to 20 10 units.

Following this, the dry lyophilized powder containing the malatedehydrogenase may be prepared for packaging in a capsule. This may beaccomplished by employing the following chemicals in the indicatedranges:

L-aspartic acid gm 220-260 Alpha-ketoglutaric acid gm 50-60 Disodiumhydrogen phosphate gm 200-600 Sodium carbonate gm 140-280 Mannitol gm800-2000 DPNH gm 6-8 Lyophilized powder units 820 (10 The alpha-asparticacid, alpha-ketoglutaric acid, disodium hydrogen phosphate and sodiumcarbonate may all be mixed together and ground into a fine powder by anysuitable means such as placing in a ball mill for about 6 to 10 hours.Following the mixing, a sample such as about milligrams of the mixturemay be dissolved in a suitable quantity of water such as 5 milliliters.The pH of this solution should be between 7.4 and 7.6. If the pH is toolow, a suitable quantity of sodium carbonate may be added to raise thepH to the desired level. If the pH is too high, additional quantities ofalpha-aspartic acid and alpha-ketoglutaric acid may be added. The ballmilling may be repeated-the powders are mixed with mannitol by ballmilling or the like, and then dried at 40 C. under vacuum in thepresence of phosphorous pentoxide.

When the foregoing powders have been blended to provide a pH in thedesired range from this point forward it is important that the powdernot be exposed to moisture. Generally, it is best that if the powder isto be exposed to the atmosphere, the relative humidity should be below15%. The DPNH and the calculated quantity of lyophilized powdercontaining malate dehydrogenase may be added to the mixture. The amountof DPNH added is such that one ten-thousandth of the resulting mixtureWhen dissolved in 3 milliliters of water will have, for convenience ofmeasurement, an optical density at 340 millimicrons of 0.8 to 1.0. Thismixture may then be ground in a ball mill.

At this point, a powder is provided that contains the enzyme malatedehydrogenase, together with the stabilizers acacia, tris-EDTA, ammoniumsulfate and ethylenediamine tetra-acetic acid so as to insure the powderbeing very stable and having a very long shelf life wherein the enzymeactivity will not deteriorate. In addition, the powder includes abufIer-disodium hydrogen phosphate and substrates alpha-aspartic acidand alpha-ketoglutaric acid that will insure the desired reactionoccurring when an assay is made. A sample of this powder is withdrawn(being careful not to introduce moisture) and tested. The tests are forthe appropriate optical density, mixing of the DPNH, and theeffectiveness of the powder to form an assay for glutamate-pyruvatetransaminase. Accordingly, this powder may be divided into a pluralityof small parts that are just large enough to form a liquid reagentsuitable for making one assay of a specimen. The entire bulk of thepowder may then be divided into units or parts which contain the desiredamount of activity. Each of these parts may then be packaged in asuitable container such as a capsule. If a pharmaceutical gelatincapsule is used, a desiccant such as an alumina pellet should be placedwith the capsule within the package. It is desirable that the same sizecapsule be employed at all times irrespective of which batch the capsuleis produced from. Accordingly, the capsule may be of a sufiicientlylarge size to insure packaging of all variations. A bulking agent suchas mannitol may then be added to each of the parts so as to bring itsvolume up to an amount that will just fill the capsule.

In order to use one of the capsules to make an assay of a serum for theglutamic oxaloacetate transaminase (GOT), a specimen of a serum or otherbiological fluid is first obtained in a suitable quantity. Followingthis, a capsule of this example is dissolved in a suitable quantity ofWater. The resultant solution will form a liquid reagent that is of justthe right size and strength to make a single assay of the serum.Accordingly, this liquid reagent may then be mixed with the specimen. Assoon as the reagent and specimen are mixed together, the followingreactions will occur:

GOT alpha-ketoglutarate-l-aspartate glutainate+oxaloacetate MDHoxaloacetate-l-DPNH malate-i-DPN Since the alpha-ketoglutarate, asparticacid and malate dehydrogenase are supplied in the capsule in sufficientamounts, the rate of the foregoing reactions will only be limited by theamount of activity of glutamic oxaloacetate transaminase or GOToriginally present. This, in turn, will cause the DPNH to be convertedto DPN in direct proportion to the amount of activity of the GOToriginally present. By placing the specimen in a suitablespectrophotometer and measuring the optical density at 340 millimicrons,the rate at which the DPNH is being converted can be determined. This,in turn, will permit the amount of activity of the GOT to be calculated.

EXAMPLE C The third example of the first embodiment is a solid reagentthat is particularly adapted to be employed for measuring or assayingthe amount of activity of aldolase present in a serum. This reagent whenfully prepared will consist of a dry mixture of the followingsubstances:

iEnzyme: triosephosphate isomerase and glyceraldehydephosphatedehydrogenase Buffer: glycine and sodium pyrophosphate Stabilizer:acacia, albumin, ethylene diamine tetra-acetic acid Substrate:fructose-1,6-diphosphate Coenzyme: DPN

Decouplcr: sodium arsenate Bulking agent: mannitol In order to preparelarge numbers of units of this reagent, the following procedure may beemployed to produce a batch of a dry reagent or assay powder that maythen be divided into small quantities and packaged into capsules.Wherever quantities are specified in this example,

they are suitable for preparing a batch that will yield on the order ofabout 10,000 capsules. It should be understood, however, that thesevalues are for illustrative purposes only and may be varied to satisfyany particular requirement.

Normally, the first step is to prepare a standard liquid assay systemwhich may be used at several different points in the procedure. Theassay liquid includes the following:

Glycine-pyrophosphate buffer, containing 998 mg. of

glycine and 181 mg. of sodium pyrophosphate deca- Distilled water tobring volume to 3 ml.

All of the foregoing liquids are mixed together to form the standardliquid assay. This solution may then be allowed to stand for arelatively short time such as several minutes. After the standard liquidassay reagent has been completed, a 1 to 400 dilution of the enzymealdolase is prepared by diluting 5 lambdas of the concentrated aldolaseBoheringer, 10 mg. per ml., to 2 milliliters with a 2 molar ammoniumsulfate solution. 5 lambdas of the diluted enzyme are then added to apart of the standard liquid. In order to obtain an accurate indication,it is necessary the enzyme be at its maximum activity at the time of thetest. Accordingly, it is desirable that this solution be preparedimmediately prior to the making of the test.

After the solution containing the enzyme has been added to the standardassay system, the resultant mixture is allowed to incubate for someinterval of time such as 5 minutes. The optical density of the solutionis then measured at 340 millimicrons by means of a suitablespectrophotometer. Thereafter, the optical density is repeatedlymeasured at this wavelength periodically for some predetermined intervalof time such as 3 minutes. By recording these readings, a referenceindicating the effectiveness or amount of activity in the standard assayliquid will be provided. Future assays in the present process may thenbe compared with this reference to determine whether they are withinacceptable ranges.

Following this, an assay powder may be prepared by mixing the followingchemicals in about the indicated ranges:

Glycine g -180 Sodium pyrophosphate g 15-35 Sodium arsenate g -250Fructose-1,6-diphosphate g 27-35 Ethylene diamine tetra-acetic acid g8-20 DPN g 10-18 Triose phosphate isomerase mg 80-200 Glyceraldehydephosphate dehydrogenase g 1.2-2.0 Albumin g 30-80 Acacia USP g 40-90 Thefirst step in preparing this powder is to mix the sodium pyrophosphateand glycine together to form a buffer powder. This mix is first groundinto a fine powder in any suitable means such as a mortar and pestle.The buffer powder may then be placed under a vacuum in the presence of amoisture absorbing agent for a suflicient period of time to insure anymoisture being removed. Following the drying, the buffer powder may befurther pulverized by placing in a ball mill for an extended period oftime. A small sample of the buffer may be 25 dissolved in water and thepH of the solution measured. The pH of this solution should be about 8.If it is not sufficiently close to this, the required quantities ofeither salt may be added to the buffer to adjust the pH to the requiredlevel and the grinding repeated.

The next step is to grind the sodium arsenate into a fine powder and addit to the glycine-sodium pyrophosphate buffer. The resultant combinationis then ball milled to form a fine powder and dried under a vacuum foran extended period of time. After this powder has been prepared, it isdesirable to test the powder to make sure that there are no inhibitorswhich may impair the operation of the assays. This determination may bemade by preparing a solution similar to the standard liquid assay.However, the just prepared powder is substituted for the first pair ofchemicals in the list for the standard assay.

The same dilution of the aldolase is then mixed with the new essayliquid and the resultant changes in the optical density at 340millimicrons measured as described in connection *with the standard. Ifthe optical density varies, the same as the reference, the preparationof the lyophilized enzymes triose phosphate isomerase and glyceraldehydephosphate dehydrogenase may proceed.

The next step will then be to dissolve the acacia into about one literof distilled water. The ethylene diamine tetra-acetic acid and thealbumin are then added to the solution and the solution thoroughlymixed. It should he noted that during the mixing a certain amount offoaming may occur. Steps should be taken to keep the foaming to aminimum. After the solution is completed, it should be placed under avacuum for a sufiicient period of time to remove any air that may stillbe trapped in the solution.

When the solution has been cleared of all air, the enzymes triosephosphate isomerase and glyceraldehyde phosphate dehydrogenase are addedto the solution. The solution should be very thoroughly mixed to insurea uniform dispersion of the enzymes throughout all portions of thesolution. Since these enzymes in this solution tend to be somewhatunstable and lose their activity, the solution should be frozen as soonas they are properly suspended in the liquid.

The frozen solution is then placed under a vacuum and all of the waterremoved therefrom.

After the enzyme mixture has been lyophilized in the foregoing manner, asample should be assayed to determine if they still have adequateactivity. This assay is made by comparing the activity of the powderwith the activity of the standard liquid assay described in thebeginning of this example. To accomplish this, a new assay solution isprepared by substituting a solution prepared from the lyophilized powderfor the enzyme solutions. The optical density should vary in the samemanner as the reference. It is desirable that this powder be storedunder refrigeration and kept as dry as possible until the process is tobe continued.

If the results of the foregoing assay are within acceptable limits, thebuffer arsenate mixture and the lyophilized powder containing theenzymes may be mixed together. Following this, thefructose-1,6-diphosphate and the DPN are added to the mixture and theentire mixture pulverized to a very fine powder by any suitable meanssuch as ball milling. The resultant powder will constitute, in bulk, adry powdered reagent containing the enzymes triosephosphate isomeraseand glyceraldehydephosphate dehydrogenase and the coenzyme DPN togetherwith stabilizers that will maintain the activity of the enzymes forextended periods of time. In order to make this solid reagent into aform that is more readily usable, it may be divided into units that areof a size suitable for assaying a single specimen. These units may thenbe enclosed in a suitable package for preventing the absorption of anymoisture. Since there may be some variations in the volumes of the unitsbetween successive batches, it may be desired to add a bulking agentsuch as mannitol to each batch that will bring the volume of each unitup to some predetermined volume. The mannitol likewise aids inconferring additional stability to the reagent. The bulked powder maythen be packaged in suitable containers such as capsules.

In order to make an assay of a serum for aldolase using one of theforegoing capsules of this example, a suitable sample of the serum orbiological fluid is first obtained. Following this, the contents of oneof the capsules is dissolved into a standard amount of water such as 3to 5 milliliters. This will produce an active liquid reagent that is ofjust the right size to make a single assay of one specimen of theforegoing size. Furthermore, the activity of this reagent will be of apredetermined level. The liquid reagent may then be mixed directly withthe specimen in a suitable test tube. As soon as the reagent andspecimen or serum are mixed together, the following reactions willoccur:

aldolase fructose-1,6-diphosphate Shydroxyacetonephosphate-l-3-glyceraldehydeph0sphate triosephosphate isomerase3-hydroxaeetonepl1osphate 3-glyceraldehydephosphateglyceraldehydephosphate 3-glycoraldehydephosphate-l-DIN dehydrogenase3-ph0sphoglycerate|-DPNH AsO;

Since the fructose-1,6-diphosphate is supplied by the capsule, it willbe present in an abundant quantity. As a result, the rate at which it isconverted directly into 3- glyceraldehyde phosphate will be limited onlyby the amount of activity of the aldolase. In addition, since thetriosephosphate isomerase is supplied in abundant quantities, the rateat which the 3-hydroxyacetonephosphate is produced and converted intothe 3-glyceraldehyde phosphate will be limited only by the amount ofactivity of the aldolase. Since the rate at which the3-glyceraldehydephosphate is produced is directly related to the amountof activity of the aldolase originally present in the specimen, the rateat which the DPN is converted to DPNH will also be determined by thealdolase. By placing the assay-reagent-specimen solution in aspectrophotometer while the foregoing reactions are progressing, therate at which the DPNH is being produced may be determined by measuringthe rate of change of the optical density of the specimen at 340millimicrons. Knowing this rate of change, by employing well-knownequations, the amount of aldolase originally present in the specimen maybe computed.

EXAMPLE D The fourth example of the first embodiment in this group is asolid reagent that is particularly adapted to be employed for measuringthe quantity of glucose present in a serum. This reagent when fullyprepared will have the following components that include the indicatedchemicals:

Enzyme: hexokinase and glucose 6 phosphate dehydrogenase Buffer:tris(hydroxy) methylaminomethane succinate Stabilizer: acacia,tris(hydroxy)methylaminomethane sulfate and ammonium sulfate Substrate:none Accelerator: insulin and magnesium sulfate Coenzymes: adenoainetriphosphate and triphosphopyridine nucleotide Bulking agent: mannitolIn order to prepare a large number of capsules or individual assayportions of this reagent, the following procedure may be employed toproduce a batch of dry reagent that may then be divided into smallquantities and packaged into capsules. Wherever quantities are specifiedin this example, they are suitable for preparing a batch that will yieldabout 10,000 capsules. It should be understood, however, that thesevalues are for illustrative purposes only and may be varied to satisfyany particular requirement.

The first step in this procedure is to prepare a suitable buffersolution. This buffer solution may be prepared by mixing the followingchemicals together in about the indicated quantities:

Grams Tris (hydroxymethyl) aminomethane 100-200 Succinic acid 30-70 Bothof these chemicals are mixed together and then ground into a fine powderby any suitable means such as placing in a ball mill for a period ofabout eight hours. The pH of the resultant mixture may then be checkedby disolving a small quantity in distilled water and measuring the pH.If the pH is not in a range of about 7.4 to 7.6, more succinic acid maybe added to lower it and more tris (hydroxymethyl) aminomethane added toraise it. It may be seen that the exact proportions of the constituentsof this buffer are determined by the pH. When the buffer is properlypowdered and the pH is within the indicated range, the mixture may bedried under vacuum at a temperature of about 50 C. in presence ofphosphorous pentoxide (P for a period of 24 hours or until the moistureis removed.

A dry lyophilized powder containing the enzymes hexokinase andglucose-6-phosphate dehydrogenase may then be prepared by mixing thefollowing chemicals in the indicated ranges:

Gum acacia gms 50-100 Tris-sulfate buffer 0.2 M, pH 7.4 to 7.6

ammonium sulfate, 1 M, pH 7.5 ml 75-150 (Adjusted with ammonia) ml 75150Insulin mg 50-750 Hexokinase mg 100 Gl-ucose-6-phosphate dehydrogenasemg 100 The tris-buifer is first prepared by making a solution containingtris (hydroxymethyl) aminomethane and adding a sufficient quantity ofsulfuric acid to make a 0.2 molar solution with a pH of 7.4 to 7.6. Theacacia, ammonium sulfate and insulin are then mixed with thetris-sulfate solution to form a homogenous solution. The resultantsolution is then placed under a vacuum for a sufficient period of timeto remove all of the air entrapped therein. The hexokinase andglucose-6-phosphate dehydrogenase are then mixed into the solution toobtain a homogeneous mixture. The solution is then frozen and placedunder a vacuum until .all of the water is removed. This will result in adry or lyophilized homogeneous powder containing the enzymes.

Following the drying thereof, the lyophilized powder may be assayed todetermine the amount of activity of the hexokinase andglucose-6-phosphate dehydrogenase enzymes by employing the followingprocedure and using the indicated quantities of the following chemicals:

Tris-succinate buffer solution (1.7 g./100 ml.) ml 1 Magnesium sulfate,0.1 molar solution ml 0.4 Triphosphopyridine nucleotide,

mg./2 ml. Water ml 0.2 Adenosine triphosphate, 9 mg./ 2 ml. water I1'I1l0.2 Lyophilized enzymes mg 10 Water ml 1.2

The tris-succinate buffer solution is first formed by dissolving 1.7grams of the buffer prepared in the first step of this example in 100milliliters of water. The rest of the listed solutions .are mixed withthe buffer and the water to form a homogeneous mixture. The opticaldensity of this solution is then measured at 340' millimicrons. Apredetermined quantity of a glucose solution of known concentration isthen added to the foregoing solution. This will Cal cause a reactionwherein the TPN will be converted to TPNH. Since the TPN will be presentin an abundant supply, the only factor which will limit the reaction isthe quantity of glucose. After the reaction has been completed, theoptical density of the solution is again measured at 340 millimicrons.The above assays are preferably made by employing several differentstandard solutions of glucose, for example, 200, 400 and 600 mg. percentof glucose.

The difference between the optical densities before and after thereaction should be equal to 0.230 times the mg. percent of the standardglucose solution divided by 100. In the event the difference is notequal to or very close this predicted value, the enzymes do not have anadequate amount of activity. However, of the changes of optical densityare equal to or very close to the predicted value, the enzymes aresufliciently active and the mixture may be prepared for packaging intothe capsules. This may be accomplished by employing the followingchemicals in the indicated ranges:

G. Tris-succinate buffer (prepared above) 100-250 Magnesium sulfate,n-hydrate 1 120 TPN 12-18 ATP 10-18 Lyophilized enzyme mixture 80-200 1An equivalent amount of magnesium may be added as the glutamate oraspartate salt.

All of these chemicals are dry solids that may be mixed directlytogether and then grounded by any suitable means such as a ball mill. Atthe completion of the grinding, the chemicals will have been properlyground to form a fine powder and have been thoroughly blended into ahomogeneous mixture. This mixture forms the assay powder and containsthe enzymes hexokinase and glucose-6-phosphate and dehydrogenasetogether with the stabilizers acacia, tris sulfate buffer and ammoniumsulfate to insure the enzymes maintaining their activity for extendedperiods of time. In addition, this assay powder also contains thecoenzymes ATP and TPN together with the buffer tris succinate to insurethe desired assay reactions occurring when an assay is being made.Accordingly, this powder may now be divided into units which .are ofjust the right size for performing a single assay of a single specimen.In order to determine the size of the units, an assay may be made todetermine the amount of activity of the enzymes in the mixture. Thisassay may be accomplished in substantially the same manner as describedabove in connection with the assaying of the lyophilized powdercontaining the enzymes.

More particularly, several small specimens of the mixture may bedissolved in suitable quantities of water to form liquid reagents. Thesame standard values of glucose (i.e., 200, 400 and 600 mg. percent ofglucose) may be mixed with the reagents and the resultant changes inoptical density measured. From the results of these tests, the amount ofactivity of the enzymes contained in the powder can be determined.

When using the assay powder in performing an assay for glucose, theresultant reaction is allowed to continue until all of the glucose hasbeen converted. The amount of time that is required for this conversionto be completed is determined by the amount of activity of the enzymes.Therefore, in order to determine the amount of activity to be includedin each capsule, it is first necessary to determine the amount of timeto be allowed for the reaction to be completed. To do this, a convenienttest period or interval is arbitrarily selected, for example, 5 minutes.Some fractional part of this interval, for example, 3 minutes, is thenselected during which the reaction is virtually completed. This willguarantee the reaction being completed at the end of the test period.The quantity of assay powder required for each capsule is thendetermined. With this quantity of activity in each capsule, it may besafely assumed that the reaction has been complete prior to the end ofthe test interval.

If it-is desired that all of the capsules be of a standard size, thecapsule size may be made larger than the largest units that may beencountered. A bulking agent such as mannitol may then be added to theassay powder so that the resultant units will just fill the capsule.

In order to make an assay of a serum for glucose using a capsule of thisexample, a specimen of the serum is first obtained in a suitablequantity for making an assay. The optical density of this specimen isfirst measured at 340 millimicrons. Following this, the contents of oneof the capsules may be dissolved in a suitable quantity of water tothereby form a liquid reagent that is of the right size and activity formaking one assay. This liquid reagent may then be combined with theserum so that the following reactions will occur:

hexokinase As these reactions occur, the glucose that was originallypresent in the serum will be converted into glucose-6-P. This, in turn,will react with the TPN to produce the TPNH. Since the capsule suppliesan excess of its chemicals, the only factor that limits the reaction isthe quantity of the glucose originally present. Therefore, the amount ofTPNH finally produced will be a function of the original quantity ofglucose.

The foregoing reaction are allowed to continue for the duration of thetest period or until they have been completed and the glucose isexhaused. After the reactions have been completed, the optical densityof the specimen is measured again at 340 millimicrons. Since the TPNHwill absorb the light at this wavelength, the difference in the opticaldensity before and after the reactions will be a result of the change inthe amount of the TPNH produced. Since the change in the quantity ofTPNH will be a direct function of the quantity of glucose originallypresent in the specimen, the change in optical density may be. employedto calculate the glucose originally present.

EXAMPLE E The last exemple of the first embodiment in this group is asolid reagent that is particularly adapted to be employed for measuringthe amount of activity of adenosine triphosphate or ATP present in abiological sample. This reagent fully prepared will consist of a drymixture of the following solid substances:

Enzyme:' hexokinase and drogenase Buffer: tris (hydroxymethyl)aminomethane succinate Stabilizer: acacia, tris (hydroxymethyl)aminomethane and ammonium sulfate Substrate: glucose Accelerator:insulin and magnesium sulfate Coenzyme: TPN

Bulking agent: mannitol In order to prepare a large number of capsulesof this reagent, the following procedure may be employed to produce abatch of dry reagent that may then be divided into small quantities andpackaged into suitable capsules. Wherever quantities are specified inthe example, they are suitable for preparing a batch that will yield onthe order of 10,000 capsules. It should be understood, however, thatwhenever desired, these quantities may be varied to satisfy anyparticular requirements.

The'first step in this procedure is to prepare a suitable buffersolution. The buffer employed is substantially identical to the trisbuffer used in Example D and consists of a mixture of tris(hydroxymethyl) aminomethane and succinic acid. These chemicals may bemixed in substaritially the same manner as described in 'Example D toproduce a dried buffer powder that when dissolved in glucose-6-phosphatedehy- 30 water wil produce a solution having a pH between 7.4 and 7.6.

A dry lyophilized powder containing the enzymes hexokinase andglucose-6-phosphate dehydrogenase may then be prepared by combining thefollowing chemicals in the indicated ranges:

Gum acacia grns 50-100 Tris-sulfate buffer 0.2 M pH 7.4 to 7.6 ammoniumsulfate 1 M pH 7.5 ml -100 (adjusted with ammonia) ml 75-100 Insulin mg50-750 Hexokinase mg 100 Glucose6-phosphate dehydrogenase mg 100 Thetris-sulfate solution is prepared first by dissolving tris(hydroxymethyl) aminomethane and adding a sufficient quantity ofsulfuric acid to make a 0.2 molar solution with a pH 7.4 to 7.6.

The acacia, ammonium sulfate and insulin are completely mixed with thetris-sulfate to form a homogenous solution. After the solution iscompletely mixed, any air entrapped therein may be removed by placingthe solution under a vacuum. The enzymes hexokinase andglucose-6-phosphate dehydrogenase are mixed into this solution. Thissolution should be thoroughly mixed to insure the enzymes beingcompletely dispersed therein. While uniformly mixed, the solution iscompetely frozen. While in the frozen state, it is placed under a vacuumuntil all of the moisture is removed. This will leave a homogeneous drypowder containing the enzymes.

This lyophilized powder may then be assayed to determine the amount ofactivity of the enzyme present by employing the following chemicals inthe indicated amounts to form a reagent:

Glucose solution 60 M/ml. (1.08 g. in

100 ml.) ml 0.5 Tris-succinate buffer solution ml 1.0 Magnesium sulfate11 hydrate 1 g./ 10 ml ml 0.4 TPN 10 mg./1 ml. water ml 0.2 Lyophilizedenzyme mixture mg 10 Water ml 1.2

The tris-succinate buffer solution is prepared by dissolving 3.4 gramsof the buffer prepared in the beginning of this example in 100milliliters of Water. Following this, the indicated quantities of therest of the solutions are combined and mixed with the indicated quantityof water. After the reagent has been completed, its optical density at340 millimicrons is measured. Following this, a standard adenosinetriphosphate solution containing 2 uM/rnl. may be added to the mixture.When this occurs, the reaction subsequently described will occur.However, the solution is allowed to stand for an adequate period of timeto insure that the reaction has been completed. After the completion ofthe reaction, the optical density at 340 millimicrons is again measured.By comparing the optical densities, the amount of change produced as aresult of the reaction can be determined.

Under the foregoing test conditions, 1 M of the ATP in ml. shouldproduce an optical density change at 340 millimicrons of 2.07.Accordingly, the 0.2 .M of the standard solution employed in this assayshould produce a change in optical density of 0.414. If such a change isproduced by the foregoing assay, the enzymes have sufficient activity tocontinue with the preparation of the described reagent.

To prepare the lyophilized powder for encapsulation, the followingchemicals may be combined in the indicated ranges:

G. Tris-succinate buffer (prepared above) 100-250 Magnesium sulfate,heptahydrate -120 T PN 12-18 Glucose, anhydrous 25-100 Lyophilizedenzyme mixture 80-200 usually 24 hours. At this point, the powder isready fordividing into suitable units and packaged into capsules. Inorder to determine the size of each unit, the activity of the powdermust first be determined. This is accomplished by dissolving a smallsample of the specimen in a suitable quantity of water such as 2.9 ml.The assay test described above may then be repeated. By measuring thechange of optical density produced by the assay reaction, the amount ofactivity of the enzymes in the powder maybe determined. When this powderis dissolved in water, it will make a reagent that will cause a reactioninvolving the adenosine triphosphate (ATP) present. This reaction willcontinue until all of the ATP has disappeared. The amount of timerequired for this to occur will be determined by the amount of activityof the enzymes present. Accordingly, in order to compute the amount ofthe assay powder to be placed in each capsule, an interval convenientfor making the test must first be chosen. For example, this may beminutes. The size of the units into which the assay powder is dividedwill then be such that the reaction will be virtually completed in somefraction of the test period, for example 3 minutes.

It is desired to have all of the capsules of some predetermined size, astandard capsule size is selected that will always accommodate thelargest unit of powder. After the size of the units is determined, asuitable quantity of a bulking and stabilizing agent such as mannitol orits equivalent may be added to bring the volume to just the right sizeto fill the standard-sized capsule.

It may thus be seen that a large number of identical capsules will beproduced that are suitable for assaying the amount of adenosinetriphosphate (ATP) present in a serum. The stabilizers tris-sulfate,acacia and ammonium sulfate will be effective to stabilize the enzymesand preserve their activity for extended period of time. Thetris-succinate buffer and the glucose substrate and TPN coenzyme willinsure the assay reaction occurring.

To use one of the capsules to assay a serum to determine the amount ofactivity of adenosine triphosphate present in a serum, a suitable sampleof the serum is first obtained. The optical density of the samplediluted with water to the same volume as the assay is first determinedat a wavelength of 340 millimicrons. The contents of one of the capsulesis then dissolved in a standard quantity of water such as 3 milliliters.This will create a liquid reagent of the correct size and activity formaking an assay of one serum sample. This liquid reagent may then becombined with the sample of serum whereby the following reactions willoccur:

hexnkinase glucose ATP glucose-64 ADP glucose-trphosphate dehydrogeuaseglucose-6P TPN G-phosgluconate TPNH As these reactions progress, the ATPwill disappear and the TPN will be converted to TPNH. Since thecornponents supplied by the reagent are in abundant supply, the onlything that will stop the reaction will be the exhausting of the ATPoriginally present. It may thus be seen that the amount of TPN convertedinto TPNH will be a function of the quantity of the ATP.

The reactions are allowed to continue for the duration of the testinterval. However, long before the expiration of this interval, thereactions will have been completed. After the reactions have beencompleted or at the end of the test interval, the optical density of thesample is measured again at 340 millimicrons. Since the TPNH will absorblight at this wavelength, the difference between the otpical densitiesbefore and after the reactions will be a result of the changes in theamount of the TPNH produced. Since the change in the quantity of TPNH isa function of the quantity of ATP originally present in the specimen,this quantity of ATP can be computetd from the change in the opticaldensity.

In the other embodiment of the invention, an assay material is providedthat is very similar to the assay material in the previously-describedembodiment. In this embodiment, the assay material is also a dry solidsuit I able for prepackaging into containers such as capsules or foilwrappers containing just the right quantity for making a single assay orpredetermined number of assays. The assay material contains all of thecomponents, except water, for making an assay. The contents of onepackage is dissolved in water to form a liquid reagent effective tocreate an enzymatic reaction of the same type created by the precedingembodiment.

The assay materials in this embodiment are particularly adapted formeasuring the amount of activity of an enzyme originally present in thespecimen. Accordingly, in this embodiment, the assay material does notnecessarily include an enzyme. However, it does include at least onesubstrate for producing the desired assay reaction and a coenzyme thatwill enter into the reaction and be converted from one form to anotherform. The coenzyme may be of the same class as in the precedingembodiment. Accordingly, the reactions produced by these assay materialsmay also be observed by measuring the optical density of thespecimenassay mixtureat the same wavelength.

In order to prepare the assay material of this embodiment, a buffermixture may be prepared first. The exact composition of the butfer willvary with the particular assay reaction to be produced. However, thebuffers are very similar to those in the first embodiment and will be ina class that includes the salts of polyvalent inorganic anions and theorganic amines together with the acids and salts thereof. By way ofexample, the salts of polyvalent inorganic anions may include at leastsodium and potassium phosphates, sodium and potassium pyrophosphates. Byway of example, the organic amines and their acids and salts may includeat least tris (hydroxymethyl) amino methane and imidazole and theirsalts such as the hydrochloride, succinate, sulfate, succinic, asparticand glutamic acids and their salts such as the sodium, potassium andlithium, glycylglycine and glycine. Normally, when these buffers havebeen fully prepared, they will be a dry powder mixture.

It is one of the purposes of the buffer to maintain the conditions suchas the pH in the reagent and specimen-- assay mixturesuitable for thedesired assay reaction to occur.

The substrates employed in this embodiment will, of course, depend uponthe particular assay reaction. However, they will normally be in a classof compounds whose chemical reactions will be specifically catalyzed bythe classes of enzymes previously referred to and whose activity isbeing measured. These substrates may also be reduced to a dry powderedform suitable for mixing with the buifer materials. The coenzymes willalso depend upon the particular assay reaction. However, the coenzyme ispreferably from the same class as in the preceding embodiment. This willinsure all of the assay reactions being effective to produce changes inthe optical density of the specimens at a standard wavelength. Moreparticularly, the coenzymes may be from the class that includes thepyridine nucleotides when in the oxidized form these coenzymes will alsohave an absorption in the region of about 340 millimicrons. The coenzymemay be in a solid form suitable for being powdered. Accordingly, thepowdered buflfers, substrates and coenzymes may all be mixed together toform the assay material.

It may thus be seen that irrespective of whether the assay to be made isto measure the amount of activityof an enzyme or not, the assay materialwill always be prepared as a dry solid material. The material may befinely powdered for prepackaging and for dissolving in a liquid such aswater to form a liquid reagent. By employing a bulking agent of the sametype described in the preceding embodiment, the various assay materialsof this embodiment may also have their volume increased to somepredetermined standard level. This will insure all of the resultantunits being of identical sizes.

It has also been found that bulking agents of this type are effective,increase the stability of the assay mixture of this embodiment. Amongother things, because of its moisture absorbing abilities, it protectsthe assay material against deterioration resulting from exposure tomoisture. Also, the bulking agents are effective to assist instabilizing substrates such as oxaloacetic acid. This acid is anunstable compound particularly when it is in a buffer solution having apH of about 7.5. By employing the present bulking agents and the methodsof preparation described herein, the acid can be made very stable and ina form that is compatible with the other components in the desiredreaction. Moreover, the oxaloacetic acid will be in a form that may beeasily handled and used. As will become apparent, the oxaloacetic acidwhen in this prepared form will be particularly suitable for use inpreparing an assay material for use in making assays for malatedehydrogenase.

It has also been found that use of the bulking agent in the preparationresults in more rapid solution of the assay mixture into water andthereby decreases the time of mixing and increases the convenience ofrapid dissolving by requiring less shaking or stirring.

It will thus be seen that this embodiment may also provide a pluralityof substantially identical packages such as metal foil packets,capsules, etc. Each of these packages will contain just a sufiicientquantity of the assay material for making a single assay of a specimenor distinct and accurate multiples of a single assay. In order to makean assay, the contents of one package is dissolved in a standard amountof water so as to form a liquid reagent. This liquid reagent is thenmixed with the specimen to produce an assay reaction. The extent of orthe rate at which this reaction occurs will be a function of the amountof activity of the enzyme present in the specimen. Since every reaction,irrespective of the particular type of assay, will involve the samecoenzyme as in the preceding embodiment, the rate at which this reactionoccurs can be measured by measuring the optical density of the specimendifferent times to determine the rate of change of the optical density.From this, it will be possible to compute the amount of activity of theenzyme originally present.

While this embodiment may be employed to assay a large number ofdifferent enzymes or other biological substances, the following examplesare illustrative of a limited number of different forms of thisembodiment.

EXAMPLE F The first example of the second embodiment is a solid assaymaterial that is particularly adapted to be employed in preparing areagent for assaying the amount of malate dehydrogenase or MDH presentin a serum. When this assay material is fully prepared, it will consistof the dry mixture of the following substances:

Buffer: sodium phosphate and potassium phosphate Substrate: oxaloaceticacid Coenzyme: DPNH Stabilizer: mannitol In order to prepare a largenumber of capsules containing this assay material, the followingprocedure may be employed to produce a batch of dry assay material thatmay be divided into small quantities and packaged into standard sizecapsules. Wherever quantities are specified, they are suitable forpreparing a batch that will yield about 10,000 capsules. It should beunderstood, however, that the amounts indicated may be varied to satisfyany particular requirements.

The assay material may be prepared by first combining the followingmaterials in the indicated amounts to form a dry mixture:

G. Sodium phosphate 200-400 Potassium phosphate 40-80 MannitolLOGO-2,000

These three ingredients are first ground into a fine powder by anysuitable means such as in a ball mill. After these materials have beenfinely ground and are thoroughly mixed, a small quantity of the powdermay be dissolved in a suitable quantity of water. The pH of theresultant solution should be in the range of about 7 .4 to 7.6. If it isnot within this range, additional quantities of one of the first twoingredients may be added to the mixture to bring the pH into the desiredrange. Although mannitol is used in the present instance, it may bereplaced with sorbitol or lactose or one of the polymers thereof havingfrom 1 to 5 hydroxyl groups per monomeric unit. The powder may then bedried under a vacuum of 0.2 mm. Hg at 25 for about 48 to 72 hours. It isalso desirable for the individual powders to be dried in this mannerbefore they are mixed together.

Following this, the coenzyme DPNH may be prepared as a fine powder andadded to the foregoing mixture. Alternatively, the coenzyme may bepowdered and added at the same time as the other powders. The quantityof the oxaloacetic acid to be added to the mixture should be carefullydetermined by the following assay. In order to make the assays, areagent is prepared by combining the indicated amounts of the followingsolutions:

Ml. 0.10 molar potassium phosphate pH 7.4 2.7 DPNH 4 mg./ml 0.1Oxaloacetic acid 1 mg./ml. 0.1 Malate dehydrogenase 0.1

The phosphate may be prepared by dissolving the phosphate to form a 0.10molar solution. The DPNH solution is formed by dissolving approximately4 milligrams of the DPNH in 1 milliliter of water. The reagent is thenprepared with a sufficient quantity of the malate dehydrogenase in 0.1ml. to cause a change of the optical density at 340 millimicrons on theorder of 0.02 per minute under the conditions of the assay. The quantityof the oxaloacetic acid added to the foregoing powder is then adjustedto produce the same rate of change in the optical density.

The amount of oxaloacetic acid that is required to be added to the assaymixture to produce the optimum rate of change in the optical density canthus be determined. This quantity of the oxaloacetic acid and the DPNHare then added to the assay mixture. The amount of DPNH that is addedwill be determined by the optical density which is desired in thespecimen and reagent mixture. Normally, this will be in the generalrange of about 0.8 CD. at 340 m After the assay material has been driedunder vacuum, a dry powder is provided that contains the substrateoxaloacetic acid and reduced diphosphopyridine nucleotide together withmannitol (as an example). As long as this powder is maintained dry, itis very stable and will have a very long shelf life. Accordingly, it maynow be divided into a plurality of small parts that are just largeenough to be mixed with water to form a liquid reagent suitable formaking a single assay of a serum. Each of these parts may then bepackaged into a suitable container such as a capsule for subsequent use.If it is desirable that the same size capsule be employed at all times,the standard size may be large enough to accept the largest quantity ofthe powder that will ever be needed. The quantity of the bulking andstabilizing agent, mannitol, added to the assay material may be selectedto bring the total volume of each part up to a size that will just fillthe capsule.

.size for making a single assay of the serum. This liquid reagent maythus be mixed with the specimen. As soon as the reagent and the specimenare mixed together, the following reaction will occur:

MDH oxaloacetic acid DPNH malic acid DPN Since the oxaloacetic acid andthe DPNH are supplied from the assay material, they will be in excess ofthe minimum amount required to complete the foregoing assay reaction.However, this reaction is dependent upon being catalyzed by the enzymemalate dehydrogenase present in the serum. Accordingly, the only factorwhich will limit the rate at which the DPNH which is converted into DPNwill be the amount of activity of the malate dehydrogenase originallypresent in the specimen. The optical density of the specimen-assaymixture is then measured at 340 millimicrons at periodic intervals. Thiswill permit the rate of change of the optical density to be compiledwhich, in turn, will permit the amount of activity of the malatedehydrogenase to be computed.

EXAMPLE G The second example of the second embodiment is a solid assaymaterial that is particularly adapted to be employed in preparing areagent for assaying the amount of lactate dehydrogenase or LDH presentsin a serum. When this assay material is fully prepared, it will consistof the dry mixture of the following substances:

Buffer: disodium hydrogen phosphate and potassium dihydrogen phosphateSubstrate: sodium pyruvate Coenzyme: DPNH Stabilizer: Mannitol G.Disodium hydrogen phosphate 200400 Potassium dihydrogen phosphate 40-80Mannitol LOGO-2,000

These three ingredients are first ground into a fine powder by anysuitable means such as in a ball mill. After these materials have beenfinely ground and are thoroughly mixed, a small quantity of the powdermay be dissolved in a suitable quantity of water. The pH of theresultant solution should be in the range of about 7.4 to 7.6. If it isnot within this range, additional quantities of one of the first twoingredinents may be added to the mixture to bring the pH into thedesired range. The buffer powder may then be dried under vacuum and inthe presence of a moisture absorbing agent such as phosphorus pentaoxide(P at a temperature of about 50 C. until all of the moisture is removed.

In addition to the buffer powder, a coenzyme and substrate powder may beprepared by combining the following materials in the indicated ranges:

DPNH z F,

In order to obtain the optimum assay reaction from the assay material,the exact amount of the sodium pyruvate is critical. Accordingly, thequantity of the sodium pyru- V3126 to be added to the mixture should becarefully determined by means of a series of assays. In order to makethe assays, a plurality of assay liquids may be prepared by combiningthe indicated amounts of the following solutions:

Phosphate buffer 1.0 g. phosphate powder above/ 10 ml. ml 2.8 DPNH 4mg./ml. mg 0.1 Serum ml 0.020

The phosphate butter may be prepared by dissolving 1 gram of thephosphate containing the powder described above in 10 milliliters ofwater. The DPNH solution is formed by dissolving approximately 4milligrams of the DPNH in 1 milliliter of water. A serum containing asuitable quantity of lactate dehydrogenase may be used as a source ofenzyme. At the same time, about 32 milligrams of the sodium pyruvate tobe employed is dissolved into about 10 milliliters of water.

After all of these solutions have been prepared, the indicatedquantities of the serum, DPNH and buffer solutions may be combined witheach other to form an assay liquid. A suitable quantity of the pyruvatesolution, for example, 0.05 milliliter is then added to the assayliquid. The optical density of the solution at 340 millimicrons ismeasured as periodic intervals and recorded. Following this, theforegoing procedure is repeated with increasing amounts of the pyruvatesolution (for example, 0.10 milliliter, 0.15 milliliter, 0.20milliliter, etc.). The optical density of the assay liquids at 340millimicrons is periodically measured and recorded. This procedure iscontinued until the exact amount of the pyruvate solution required toproduce the maximum rate of change of the optical density at 340millimicrons is determined.

The amount of sodium pyruvate that is required to be added to the assaymixture to produce the optimum rate of change in the optical density canthen be determined. This quality of the pyruvate and the DPNH are thenadded to the assay mixture. The amount of DPNH that is added will bedetermined by the optical density which is desired in thespecimen-reagent mixture. Normally, this will be in the general range ofabout 0.8 CD. at 340 m After the sodium pyruvate and -DNPH have beenadded to the previously prepared mixture, the resultant mixture may beground and pulverized by any suitable means such as ball mill to formthe powdered assay material. Following this, the assay material may bedried under vacuum.

At this point, a dry powder is provided that contains the substratesodium pyruvate and reduced diphosphopyridine nucleotide together withmannitol (as an example). As long as this powder is maintained dry, itis very stable and will have a very long shelf life. Accordingly, it maynow be divided into a plurality of small parts that are just largeenough to be mixed with water to form a liquid reagent suitable formaking a single assay of a serum. Each of these parts may then bepackaged into a suitable container such as a capsule for subsequent use.If it is desirable that the same size capsule be employed at all times,the standard size may be large enough to accept the largest quantity ofthe powder that will ever be needed. The quantity of the bulking andstabilizing agent, mannitol, added to the assay material may be selectedto bring the total volume of each part up to a size that will just fillthe capsule.

In order to use one of the capsules to make an assay of the amount ofactivity of the lactate dehydrogenase present in a serum, a specimen ofthe serum is first octained. Following this, the assay material in oneof the capsules of this example is dissolved in a suitable quantity ofwater. This will form a liquid reagent having the right size for makinga single assay of the serum. This liquid 37 reagent may thus be mixedwith the specimen. As soon as the reagent and the specimen are m'nredtogether, the following reaction will occur:

LDH pyruvate DPNH laetate DPN This reaction is dependent upon beingcatalyzed by the enzyme lactate dehydrogenase present in the serum.Accordingly, the only factor which will limit the amount of the DPNHwhich is converted into DPN under the test conditions will be the amountof activity of the lactate dehydrogenase originally present in thespecimen.

EXAMPLE H This exemplifies a solid assay material that is particularlyadapted to be employed in preparing a reagent for assaying the amount ofalpha-hydroxybutyrate dehydrogenase or HBDH present in a serum. Whenthis assay material is fully prepared, it will consist of the drymixture of the following substances:

buffer: disodium hydrogen phosphate and potassium dihydrogen phosphateSubstrate: sodium alpha-ketobutyrate Coenzyme: DPNH Stabilizer: mannitolGrams Disodium hydrogen phosphate 200-400 Potassium dihydrogen phosphate40-80 Mannitol LOGO-2,000

The first two ingredients are first ground into a fine powder by anysuitable means such as in a ball mill. After these materials have beenfinely ground and are thoroughly mixed, a small quantity of the powdermay be dissolved in a suitable quantity of water. The pH of theresultant solution should be in the range of about 7.4 to 7.6. If it isnot within this range, additional quantities of one of the first twoingredients may be added to the mixture to bring the pH into the desiredrange. The buffer powder may then be dried under vacuum and in thepresence of a moisture absorbing agent such as phosphorus pentoxide (Pat a temperature of about C. for two days.

In this example, the buffer consists of a mixture of disodium hydrogenphosphate and potassium dihydrogen phosphate; however, equivalentbuffers such as those composed of alkali rnetal phosphate ortris(hydroxymethyl) aminomethane and succinic acid may be employed. Thebuffer used should be an anhydrous powder before dissolving in water atwhich time the resultant solution should have a pH in the range of 7.4to 7.6.

After the first two ingredients have been mixed and pulverized,vacuum-dried mannitol is mixed with them. The resulting buifer-mannitolpowder is kept dry throughout the succeeding steps of processing.

In addition, to the bulfer-mannitol powder, a coenzyme and substratepowder may be prepared by combining the following materials in theindicated ranges:

G. Sodium alpha-ketobutyrate 25-50 DPNH 5-7.5

In order to obtain the optimum assay reaction from the assay material,the exact amount of the sodium alpha- 38 ketobutyrate is critical.Accordingly, the quantity of the sodium alpha-ketobutyrate to be addedto the mixture should be carefully determined by means of a series ofassays. In order to make the assays, a plurality of assay liquids may beprepared by combining the indicated amounts of the following solutions:

Phosphate buffer 1.0 g. bulfer-mannitol above/ 1 0 ml 2.8 DPNH 4 mg./ml0.1

Serum 0.020

The phosphate buffer may be prepared by dissolving 1 gram of thephosphate containing the powder described above in 10 milliliters ofwater. The DPNH solution is formed by dissolving approximately 4milligrams of the DPNH in 1 milliliter of water. A serum containing asuitable quantity of lactate dehydrogenase may be used as a source ofenzyme. As the same time, about 320 milligrams of the sodiumalphaketobutyrate to be employed is dissolved into about 10 millilitersof water.

After all of these solutions have been prepared, the indicatedquantities of the serum, DPNH and buffer solutions may be combined witheach other to form an assay liquid. A suitable quantity of thealpha-ketobutyrate solution, for example, 0.05 milliliter is then addedto the assay liquid. The optical density of the solution at 340millimicrons is measured at periodic intervals and recorded. Followingthis, the foregoing procedure is repeated with increasing amounts of theketo butyrate solution (for example, 0.10 milliliter 0.15 milliliters,0.20 milliliter, etc.). The optical density of the assay liquids at 340millimicrons is periodically measured and recorded. This procedure iscontinued until the exact amount of the ketobutyrate solution requiredto produce the maximum rate of change of the optical density at 340millimicrons is determined.

The amount of sodium alpha-ketobutyrate that is required to be added tothe assay mixture to produce the optimum rate of change in the opticaldensity can then be determined. This quantity of the sodiumalpha-ketobutyrate and the DPNH are then dried under vacuum at roomtemperature for 12 to 24 hours and added to the buifer-mannitol mixture.The amount of DPNH that is added will be determined by the opticaldensity which is desired in the specimen. Normally, this will be in thegeneral range of about 0.8. After the sodium pyruvate and DPNH have beenadded to the previously prepared mixture, the resultant mixture may beground and pulverized by any suitable means (which prevents exposure toexcess moisture) such as a ball mill form the thoroughly mixed powderedassay material. Following this, the assay material may be dried undervacuum.

At this point, a dry powder is provided that contains the substratesodium alpha-ketobutyrate and coenzyme diphosphopyridine nucleotide(reduced) together with mannitol (as an example). As long as this powderis maintained dry, it is very stable and will have a very long shelflife. Accordingly, it may now be divided into a plurality of small partsthat are just large enough to be mixed with water to form a liquidreagent suitable for making a single assay of a serum. Each of theseparts may then be packaged into a suitable container such as a capsulefor subsequent use. It it is desirable that the same size capsule beemployed at all times, the standard size may be large enough to acceptthe largest quantity of the powder that will ever be needed. Thequantity of the buliking and stabilizing agent, mannitol, added to 0118essay material may be selected to bring the total volume of each part upto a size that will just fill the capsule.

Mannitol has been found to be the preferably bulkingstabilizing agent;however, any equivalent material which dissolves readily, does notinterfere with the chemical reaction of the assay, is not hygroscopicand contains negligible moisture, may be an equivalent contemplated bythe inventor for carrying out his invention.

Equivalent materials are typically polyhydric substances and polymers ofsaid polyhydric substances with from 1 to 5 hydroxyl groups permonomeric unit.

In order to use one of the capsules to make an assay of the amount ofactivity of the alpha-hydroxybutyrate dehydrogenase present in a serum,a specimen of the serum is first obtained. Following this, the assaymaterial in one of the capsules of this example is dissolved in asuitable quantity of water. This will form a liquid reagent having theright size for making a single assay of the serum. This liquid reagentmay thus be mixed with the specimen. As soon as the reagent and thespecimen are mixed together, the following reaction will occur:

HDBH alpha-ketobutyrate l DPN H alpha-hydroxybutyrate DPN Since thesodium alpha-ketobutyrate and the DPNH are supplied from the assaymaterial, this reaction is dependent upon being catalyzed by the enzymealpha-hydroxybutyrate dehydrogenase present in the serum. Accordingly,the only factor which will limit the amount of the DPNH which isconverted into DPN under the test conditions will be the amount ofactivity of the alpha-hydroxybutyrate dehydrogenase originally presentin the specimen.

EXAMPLE I The fourth example of the second embodiment is a solid assaymaterial that is particularly adapted for making a liquid reagent thatmay be employed for assaying the amount of activity ofglucose-6-phosphate dehydrogenase. This material when fully preparedwill consist of a dry mixture of the following:

Buffer: tris-succinate Substrate: glucose-fi-phosphate, sodium saltCoenzyme: TPN

Accelerator: magnesium sulfate, glutamate or aspartate Stabilizer:mannitol In order to prepare a large number of capsules of this assaymaterial, the following procedure may be employed to produce a batch ofa dry reagent or assay powder that may be divided into small quantitiesand packaged into capsules. Wherever quantities are specified in thisexample, they are suitable for preparing a batch that will yield about10,000 capsules. It should be noted that the values are for illustrativepurposes only and may be varied to satisfy any particular requirement.

Normally, the first step in making an assay material of this form is toprepare a buffer. In the present example,

the mixture of the buffer includes the following materials The tris(hydroxymethyl) aminomethane and the succinic acid are first mixedtogether and ground into a fine powder in the same manner as describedbefore. This Will provide a tris-succinate buffer that is in the form ofa fine powder, which when dissolved in water will form a solution havinga pH of 7.4 to 7.6.

Following the preparation of the tris-succinate buffer, the magnesiumsulfate may be added thereto (an equivalent amount of magnesium as itsglutamate or aspartate salt may be used). This will be in solid form andaccordingly may be mixed directly with the buffer and the resultantmixture ground into a fine powder.

It is desirable to make sure that there are no inhibitors present in thesulfate-bufier which might interfere with the final assays. To do this,the following solutions are prepared in the indicated manner:

The first solution is formed by dissolving 10 milligrams ofglucose-6-phosphate sodium salt in 10 milliliters of water (10, molartest).

The second solution is also formed by dissolving 11.5

milligrams of triphosphopyridine nucleotide in 1 milliliter of water(1.5 molar test).

A third solution is prepared by dissolving 430 milligrams of the mixtureof the magnesium sulfate and trissuccinate buffer in 1.8 milliliters ofwater.

A fourth solution is also prepared that includes tris (hydroxymethyl)aminomethane, succinic acid and magnesium sulfate which are obtainedfrom sources differing from those used in preparing the magnesium-buffermixture, but are dissolved to form a solution that is the equivalent ofthe third solution. A first test solution is formed by combining 0.1milliliter of the first or glucose- 6-phosphate solution, 0.1 milliliterof the second or triphosphopyridine solution, 1.8 milliliters of thethird or buffer mixture and enough water to make the final volume equalto 2.9 millilters.

A second test solution is formed by combining the buifer solutionobtained from the original or step 1, 0.1 milliliter of theglucose-6-phosphate solution, 0.1 milliliter of the second ortriphosphopyridine nucleotide solution, 1.8 milliliters of the fourth orequivalent of the prepared buffer and sufficient water to make a totalof 2.9 milliliters.

After the two solutions are prepared, 0.1 milliliter of a solution ofglucose-6-phosphate dehydrogenase is placed in one of the testsolutions. The changes in the optical density of the solution occurringat 340 millimicrons are measured. The same amount of glucose-6-phosphatedehydrogenase (0.1 milliliter) is then added to the second testsolution. The changes in the optical density of this solution are thenmeasured. If the changes for the two solutions are of the same, themagnesium-buffer mixture contains no inhibitors and the assay materialmay be prepared.

The assay material may be prepared -by mixing the following materialstogether in the indicated amounts:

G. Magnesium-tris succinate buffer 400-600 Glucose-6-phosphate 27-33Triphosphopyridine nucleotide 10-13 The indicated quantities of thechemicals are mixed together and placed in a ball mill until they areground into a fine powder.

At this point, a dry powder is provided that contains the substrate andcoenzyme that are to react with each other to form the assay reaction.This powder is very stable and will have a long shelf life. Accordingly,it may be divided into a plurality of small parts that are just largeenough to be mixed with water to form a liquid reagent suitable formaking a single assay of a serum. Each of these parts may then bepackaged into a suitable container such as a capsule for subsequent use.If it is desirable that the same size capsule be employed at all times,the standard size may be large enough to accept the largest quantity ofthe powder that will ever be needed. A bulking and stabilizing agentsuch as mannitol is added so as to increase the total volume and improvethe stability.

In order to use one of the capsules to make an assay of a serum todetermine the amount of activity of glucose- 6-phosphate dehydrogenase,a specimen of the serum is first obtained. Following this, the assaymaterial in one of the capsules of this example is dissolved in asuitable quantity of water. This will form a liquid reagent having theright size for making a single assay of the serum. This liquid reagentmay thus be mixed with the specimen. As soon as the reagent and thespecimen are mixed together, the following reaction will occur:

glucose-6-phosphate dehydrogenase glucose-dphosphate l- TPNfi-phospho-gluconate l TPNH

